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Substantial variability exists in collateral density and ischemia-induced collateral growth among species. To begin to probe the underlying mechanisms, which are unknown, we characterized two mouse strains with marked differences in both parameters. Immediately after femoral artery ligation, collateral and foot perfusion were lower in BALB/c than C57BL/6 (P < 0.05 here and below), suggesting fewer pre-existing collaterals. This was confirmed with angiography and immunohistochemistry (∼35% fewer collaterals in the BALB/c's thigh). Recovery of hindlimb perfusion was attenuated in BALB/c, in association with 54% less collateral remodeling, reduced angiogenesis, greater ischemia, and more impaired hindlimb use. Densities of CD45+ and CD4+ leukocytes around collaterals increased similarly, but TNF-α expression was 50% lower in BALB/c, which may contribute to reduced collateral remodeling. In normal tissues, compared with C57BL/6, BALB/c exhibit an altered arterial branching pattern and, like skeletal muscle above, have 30% fewer collaterals in intestine and, remarkably, almost none in pial circulation, resulting in greatly impaired perfusion after cerebral artery occlusion. Ischemic induction of VEGF-A was attenuated in BALB/c. Analysis of a C57BL/6 × BALB/c recombinant inbred strain dataset identified a quantitative trait locus for VEGF-A mRNA abundance at or near the Vegfa locus that associates with lower expression in BALB/c. This suggests a cis-acting polymorphism in the Vegfa gene in BALB/c could contribute to reduced VEGF-A expression and, in turn, the above deficiencies in this strain. These findings suggest these strains offer a model to investigate genetic determinants of collateral formation and growth in ischemia.
- pre-existing collateral network
- collateral development and arteriogenesis
- cerebral circulation
- vascular endothelial growth factor and mouse strains
- expression quantitative trait loci
understanding the adaptive processes of collateral formation and growth (arteriogenesis) and capillary sprouting (angiogenesis) in ischemia is important in treating occlusive vascular diseases of the heart, brain, and peripheral limbs. Besides the arterial-capillary-venous and lymphatic circulations, collaterals constitute a "third circulation" comprising vessels with unique properties that are present in most tissues. Collaterals are rare arteriole-to-arteriole anastomoses ∼10–30 μm in diameter that interconnect adjacent major arterial trees. Because collaterals lack a significant pressure drop over their length, collateral endothelial cells are normally exposed to little or no flow-dependent shear stress (21). However, critical narrowing or occlusion of the main artery supplying one of the trees causes a pressure drop across the collaterals. This increases flow/shear stress inducing them to outwardly (positively) remodel into large caliber "endogenous bypass vessels."
Collateral growth has greater potential for restoring oxygen delivery to a tissue after artery occlusion than angiogenesis, which can only improve distribution of flow supplied by collaterals. However, compared with angiogenesis much less is known about mechanisms that mediate arteriogenesis. Moreover, nothing is known about how collaterals develop in normal tissues. Collateral density varies widely among species (21). For example, coronary collaterals are abundant in guinea pigs and sparse in swine, resulting in minimal damage in guinea pigs and severe infarctions in swine following occlusion of a major branch of the left coronary artery (44). Similarly, findings in a recent study (62) suggest that collateral density may also vary widely among humans. Coronary collateral conductance was found to exhibit up to a 10-fold variation in a group of 60 normal human subjects. There is also evidence that collateral remodeling varies among humans with diabetes, dyslipidemia, and hypertension. It is known from animal studies that these diseases impair the remodeling process (2, 50, 58).
Although these differences suggest that variation in collateral number and capacity for remodeling may have a genetic component, there is little data addressing this possibility. Recently, BALB/c mice were reported to exhibit significantly reduced recovery of blood flow after femoral artery ligation compared with C57BL/6 (17, 45). Provocative evidence has also been reported suggesting that BALB/c have fewer pre-existing collaterals (22). Unfortunately, these studies were not able to differentiate potential contributions of differences in capillary density, caliber of pre-existing collaterals, collateral remodeling, or angiogenesis to the impairment in the BALB/c strain. Nevertheless, if these two strains differ significantly in collateral density or collateral remodeling, this would provide a powerful model to investigate genetic determinants of normal collateral development, as well as collateral growth in ischemia. Therefore, the purpose of this study was to characterize in detail the arteriogenic and angiogenic response to ischemia in these strains and to investigate potential mechanisms. In addition to substantial differences in collateral remodeling and angiogenesis, we report that these strains have a profound difference in collateral density in multiple tissues. These differences result in large differences in flow impairment after arterial occlusion. Significant variation exists between the strains in arterial tree structure and VEGF-A expression, suggesting that this morphogen/cytokine may be important in specifying collateral density. In addition, we analyzed a genome-wide microarray expression database generated for a recombinant inbred set of mice derived from a cross of BALB/c and C57BL/6 mice. Multiple quantitative trait loci were identified on MMU17, near the Vegfa locus, that associate with differences in expression in the BALB/c vs. C57BL/6 genomes of VEGF-A and several genes that drive its expression or are driven by downstream VEGF-A signaling.
Hindlimb ischemia model.
We purchased 11- to 12-wk-old male mice (C57BL/6 and BALB/c) from Charles River Laboratory, (Wilmington, MA), Taconic Farms (Germantown, NY), and The Jackson Laboratory (Bar Harbor, ME) to minimize any potential effect on results of vendor-specific genetic differences between the strains. No differences were detected among mice of the same strain from the three vendors. The femoral artery was ligated proximal to the genu artery and distal to the origin of both the lateral caudal femoral and superficial epigastric arteries (the latter was also ligated) as described previously (5). All procedures were approved by the University of North Carolina Institutional Animal Care and Use Committee.
Laser Doppler perfusion imaging.
C57BL/6 (26.9 ± 0.3 g, n = 10) and BALB/c (26.0 ± 0.4 g, n = 10) were used. Hindlimb perfusion was assessed noninvasively in the ventral adductor thigh region and plantar foot before, immediately after, and 3, 7, 14 and 21 days after femoral ligation by scanning laser-Doppler [model LDI2-IR modified for high resolution (5); Moor Instruments, Wilmington, DE]. The hindquarters were placed in a heated positioning mold during scanning to minimize variation in positioning during scanning. Doppler perfusion of the ventral adductor and plantar foot was assessed within anatomically defined regions of interest (ROIs; High Resolution, version 5.0, Moor Instruments). The ROI for the adductor was drawn to obtain perfusion within the collateral zone [CZ, collateral-containing region between the lateral caudal femoral artery (LCFA) and saphenous artery (SA)] (Fig. 1A). The ROI for the plantar foot consisted of the hind paw margins (Fig. 1B). Procedures for animal preparation, scanning, and ROI construction have been described in detail previously (5). All ROIs were drawn by an investigator blinded to animal genotype (and for other analyses, below, where possible). The average velocity in a ROI was normalized to the area of the ROI. This was done because ROI construction differed for the same animal on different imaging days as a result of unavoidable variation in animal positioning during scanning.
Histological analysis.
Tissue preparation, histological procedures, and measurements have been detailed previously (5). Lumen diameters of collaterals in the anterior and posterior gracilis muscles were measured in the collateral midzone, angiogenesis was assessed in the adductor (semimembranosus and caudal femoralis) and calf (gastrocnemius), and α-smooth muscle actin-positive (α-SMA+) vessel density was determined in the CZ of the semimembranosus muscle in the adductor. Leukocyte and T-helper cell densities were determined around pre-existing and remodeled collaterals in the gracilis muscles. Positive cells were labeled with a rat anti-mouse CD45 antibody (pan-leukocyte marker, Ly-1; 1:200; BD Biosciences, Pharmingen, Boston, MA) or rat anti-mouse CD4 antibody (T-helper, GK1.5:sc-13573; 1:200; Santa Cruz Biotechnology, Santa Cruz, CA), biotinylated mouse anti-rat secondary antibody (1:100, Vectastain ABC), and diaminobenzidine. Sections were lightly counterstained with Mayer's hematoxylin solution. Cells with a blue nucleus surrounded by a brown reaction product on their surface were counted. Counts were normalized to area (∼260 × 340 μm) within the field of view.
In vivo assessment of ischemia and function in the hindlimb.
At 3, 7, 14, and 21 days after femoral artery ligation, animals were individually inspected for foot appearance score [index of ischemia: 0, normal; 1–5, cyanosis or loss of nail(s), where the score is dependent on the number of nails affected; 6–10, partial or complete atrophy of digit(s), where the score reflects number of digits affected; 11, partial atrophy of forefoot]. Hindlimb use scores (index of muscle function) were obtained (53): 0, normal; 1, no toe flexion; 2, no plantar flexion; 3, dragging foot.
Postmortem angiography and analysis of tree structure in skeletal muscle.
In a separate set of experiments, mice were perfusion-cleared, heparinized, maximally dilated (10 mg/ml adenosine and 4 mg/ml papaverine, freshly prepared), fixed with 4% paraformaldehyde (PFA) at 100 mmHg, and placed in a positioning mold, as described in detail previously (4). The vasculature was injected with barium sulfate to provide X-ray arteriograms, as described previously. Films were digitized, and a Rentrop-like line analysis was performed by counting detectable arteries intersecting: 1) a vertical line beginning at the midpoint between the proximal and distal ligations of the femoral artery and extending through the CZ to the posterior edge of the thigh (index of pre-existing collateral number in the acutely ligated limb and remodeled collaterals in the day 7 and 21 ligated limbs), 2) a vertical line beginning at the medial epicondyle of the femur and extending to the edge of the calf (index of conductance of pre-existing collateral network), 3) a vertical line extending from the lesser trochanter of the femur to the posterior edge of the thigh of the acutely ligated leg (index of second-order arteries branching laterally from the profundus artery), and 4) a horizontal line connecting the iliac crest to the lateral edge of the abdominal wall (estimate of second-order arteries branching caudally from the superior epigastric artery). In ROI in the thigh and calf, pixel intensity of barium in each artery (index of amount of arterial filling) was determined in an anatomically defined area (area intensity) with ImageJ (National Institutes of Health). The area-intensity values from the chronically ligated limb were normalized to the acutely ligated limb.
Determination of collateral number and tree structure in the pial and intestinal circulations.
A separate set of animals was cannulated via the descending abdominal aorta, heparinized, perfusion-cleared, and maximally dilated (10 mg/ml adenosine and 4 mg/ml papaverine). Animals, maintained at 37°C, were then recannulated in the ascending abdominal aorta and perfused with a mixture of 45% (wt/vol) barium sulfate, 12% (wt/vol) gelatin, and 1 mg/ml fluorescein isothiocyanate dextran as above (FITC-dextran-180, T-1287; Sigma-Aldrich). This formulation of FITC-dextran-barium gel results in only a dilute amount of FITC-dextran crossing the capillaries, giving a faint image of venules (Figs. 4, A and C, and 5D). Animals were then chilled on ice. The vasculature was imaged with a Leica MZ16FA microscope (Leica Microsystems, Bannockburn, IL). The number of collaterals connecting the middle cerebral and anterior cerebral arterial (ACA) circulations of both hemispheres was counted. Pial collateral diameter was determined from images focused in the plane of the collaterals (ImageJ). The relative areas supplied by the anterior, middle, and posterior artery trees were determined from images of the dorsally visible cortical area overlain by each cerebral artery tree (this flat field does not take into account the effect of the curvature of the brain).
In animals filled with the FITC-dextran-barium gel solution, images of the intestinal circulation were acquired after pinning the intestine out. The number of collaterals interconnecting second-order arteries in the intestine was determined.
Functional assessment of middle cerebral artery collaterals.
In a separate group, mice were perfused, cleared, and vasodilated as described above. The vasculature was perfusion-fixed with 2% PFA. The descending thoracic aorta was cannulated in a retrograde manner, and the ascending thoracic aorta was ligated. The pial circulation was then exposed, and the left and right middle cerebral artery (MCA) trunks were resected after ligation. A solution of Evans blue dye (4 mg/ml) and albumin (10 mg/ml) in 100 mmol/l sodium phosphate buffer (pH 7.4) was infused into the aorta at 60 mmHg of constant pressure. The time required to fill the second-order branches of the MCA was recorded using digital video microscopy.
Expression of TNF-α and VEGF-A.
Adductor and gastrocnemius were harvested, frozen in liquid nitrogen, and stored at −80°C. Tissue was powdered with a tissue pulverizer cooled in liquid nitrogen and homogenized in TRIzol Reagent (Invitrogen, Carlsbad, CA). Total RNA was extracted according to the manufacturer's protocol. Genomic DNA was removed using a TURBO DNA-free Kit (Ambion, Austin, TX). cDNA was prepared from 1 μg of total RNA using Superscript III reverse transcriptase (Invitrogen). An equal amount of total RNA was included as a no-RT control for each tissue sample.
Samples for real-time PCR included cDNAs (1:50) in triplicate and the respective no-RT control, along with a no-template control for each gene analyzed. Real-time PCR for the expression of TNF-α was performed on a Mx3000P Real-Time PCR machine (Stratagene, La Jolla, CA) using the Absolute QPCR mix (ABgene, Rochester, NY). TNF-α forward primer was 5′-CTGTCTACTGAACTTCGGGGTGAT-3′ and TNF-α reverse primer was 5′-GGTCTGGGCCATAGAACTGATG-3′. The probe for TNF-α detection was 5′-FAM-ATGAGAAGTTCCCAAATGGCCTCCCTC-TAMRA-3′. A GAPDH primer/probe (#4308313; Applied Biosystems, Foster City, CA) was used as an internal control for all samples. The PCR cycling parameters were 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 60 s.
Real-time PCR for the expression of VEGF-A isoforms was performed on the Rotor-Gene 3000 (Corbett Life Science, Sydney Australia) using SYBR Green chemistry (SYBR Green JumpStart Taq ReadyMix, S4438, Sigma-Aldrich). Murine VEGF-A was amplified using a forward primer common to all VEGF-A isoforms (exon3F) and reverse primers specific to each isoform. Cyclophilin A was used as a control. Primers were designed using VectorNTI (Invitrogen, Carlsbad, CA). The following oligonucleotides were purchased from Invitrogen: VEGF-A common forward primer, VEGF-A-exon3F (5′-ATCTTCAAGCCGTCCTGTGTGC-3′), VEGF-A isoform specific reverse primers: VEGF-A-120R (5′-TTGGCTTGTCACATTTTTCTGG-3′), VEGF-A-164R (5′-CAAGGCTCACAGTGATTTTCTGG-3′), VEGF-A-188R (5′-ATCTTCAAGCCGTCC-TGTGTGC-3′), cyclophilin A forward primer (5′-CAGACGCCACTGTCGCTTT-3′), and cyclophilin A reverse primer (5′-TGTCTTTGGAACTTTGTCTGCAA-3′). The PCR cycling parameters were 95°C for 5 min, 45 cycles of 95°C for 10 s, 60°C for 20 s, and 72°C for 25 s. Fluorescence acquisition was performed at the end of each cycle. Specificity of the PCR was verified as a single band visualized with 4% agarose gel electrophoresis and melt curve analysis.
Fluorescence threshold was set and the cycle at which each sample crossed the threshold (Ct) was recorded in triplicate and averaged. mRNA was quantified using the 2−ΔΔCt mathematical model, where ΔΔCt = ΔCtBALB/c − ΔCtC57BL/6(ΔCt = CtGene of Interest − CtHousekeeping gene).
Expression quantitative trait loci mapping.
Interval mapping was performed using WebQTL (GeneNetwork; http://genenetwork.org) (60) on an expression database for hippocampus mRNA (Hippocampus consortium CXB M430v2). This database was generated from a recombinant inbred set of 13 genetically distinct strains derived from the F2 cross of BALB/c and C57BL/6 mice (CXB, 7, 14, 61) that has been genetically mapped in fine detail (MIT markers; 52). Expression levels of genes of interest obtained by microarray analysis (Affymetrix, see Supplemental Table S1 for probe sets)1 of the 13 strains were mapped. Logarithm of odds (LOD) scores of ≥3.0 were taken as suggesting a genetic linkage between expression level and the marker locus for the two progenitor strain genotypes. A cis-acting expression quantitative trait locus (eQTL) was defined as an LOD ≥3.0 that mapped within ∼10 cM of the gene's physical locus (13). Allele effects of each eQTL were obtained to estimate the phenotype of the gene of interest for the two progenitor strains.
All data are reported as means ± SE. Statistical significance (P < 0.05) was determined by two-way ANOVA followed by Dunn-Bonferroni corrected t-test, paired t-test for within animal comparisons, and unpaired t-tests for group comparisons. Parametric analysis was conducted on all data except capillary density and capillary number-to-muscle fiber ratio, which were subjected to nonparametric analysis. For qRT-PCR, Student's t-test was used to determine if the fold change was significantly different from 1, which corresponds to the null hypothesis that there is no difference in TNF-α or VEGF-A expression (i.e., ΔCtC57BL/6 = ΔCtBALB/c, therefore 2−ΔΔCt = 2° = 1).
BALB/c mice have reduced hindlimb perfusion immediately after femoral artery ligation and impaired recovery over time.
The hindlimb has a small population of collaterals in the middle of the thigh (CZ). These interconnect several distal arterioles of the profunda (deep femoral artery) and LCFA with the popliteal and SA trees. We used noninvasive high-resolution infrared laser Doppler imaging (∼2 mm sampling depth) (5) to measure perfusion in the CZ and plantar foot of C57BL/6 and BALB/c mice after femoral artery ligation (Fig. 1). Doppler perfusion in the CZ before ligation reflects primarily capillary perfusion supplied either by the SA or LCFA, since collaterals in normal tissues exhibit little or no net flow (21). In contrast, perfusion immediately after ligation reflects both capillary flow and collateral perfusion induced by the pressure drop caused by ligation. Perfusion in the CZ dropped immediately after ligation in BALB/c but was sustained in C57BL/6 (Fig. 1C), suggesting fewer or narrower collaterals in BALB/c mice. The contribution to the perfusion signal from the collaterals is affected by their volume flow, diameter, and velocity. As collateral diameter remodels outward, collateral velocity may vary inversely, depending on volume flow. Since collateral diameter (dilated, fixed) was determined at the end of the study by histology (day 21, discussed below), only inferences can be made regarding how the factors determining adductor perfusion vary over time. Perfusion in the adductor region immediately after femoral artery ligation is influenced by autoregulatory dilation of downstream resistance vessels and flow-mediated dilation of feed arteries upstream of the collaterals. Increased velocity in the adductor collaterals immediately after ligation favors relaxation of collateral smooth muscle tone. Since morphometric analysis revealed no differences in lumen diameter under maximally dilated conditions in the two strains before ligation (Fig. 2A, discussed below), the initial perfusion deficit in the BALB/c adductor region, compared with C57BL/6 mice (Fig. 1C), is consistent with them having fewer pre-existing collaterals compared with C57BL/6 mice (X-ray angiography findings in Fig. 3 support this, discussed below).
During the first week after ligation, C57BL/6 showed a pronounced increase, whereas BALB/c exhibited a delayed increase (Fig. 1C). This delayed rise is also consistent with the smaller number of vessels we detect in the collateral zone in BALB/c mice 7 days after ligation (Fig. 3, B and D; discussed below) and the smaller amount of collateral outward remodeling presumably achieved by that time given that collateral diameters of BALB/c are smaller on day 21 (Fig. 2A, discussed below). The progressive rise in adductor perfusion in BALB/c mice between 3 and 21 days is consistent with less remodeling of fewer collaterals, resulting in higher collateral velocities as overall leg blood flow (indicated by plantar perfusion in Fig. 1D) increases over 3 wk as collateral remodeling and angiogenesis in the ischemic lower leg progress. By contrast, the decline in adductor perfusion in the C57BL/6 mice between day 7 and day 21 is consistent with the higher overall leg flow at all time points (Fig. 1C) conducted through a larger number of collaterals in parallel (Fig. 3, B and D; discussed below) that undergo greater outward remodeling (Figs. 2A and 3, B and D; discussed below). This results in declining velocity in the individual collaterals as indicated by the declining perfusion flux values in Fig. 1C. Perfusion in the plantar foot, which correlates with hindlimb collateral conductance (45), dropped more in BALB/c immediately after ligation and exhibited a lower recovery, compared with C57BL/6 (Fig. 1D). The greater drop in plantar perfusion immediately after ligation is also consistent with less collateral capacity in BALB/c mice.
Our Doppler measurements, like those of others, were obtained at the prevailing level of vascular tone because it not possible to obtain noninvasive perfusion measurements of the mouse hindlimb during maximally dilated conditions. An attempt to determine hindlimb vascular tone noninvasively in the mouse by using whole hind leg "cuff" reactive hyperemia was reported previously (22), but the results were inconclusive possibly because the time required for Doppler scanning may have limited detection of the full reactive hyperemia curve.
Arteriogenesis and angiogenesis are inhibited in BALB/c mice.
Since arterial pressure does not differ in isoflurane-anesthetized (65) [and conscious (19)] C57BL/6 and BALB/c mice, the above differences do not reflect lower arterial pressure in BALB/c. Rather, the lower perfusion in the CZ and foot immediately after ligation suggest an anatomic difference, i.e., that the BALB/c hindlimb has fewer pre-existing collaterals, narrower collaterals, and/or fewer capillaries. Differences in perfusion during the subsequent 3 wk suggest a mechanistic difference in BALB/c, i.e., deficient collateral remodeling and/or angiogenesis. To differentiate among these possibilities, we next asked if diameter of collaterals in the CZ of the anterior and posterior gracilis muscles (whose perfusion are sampled by the above Doppler system) and capillary densities in the CZ of the adductor thigh muscles and in the gastrocnemius are smaller before ligation in BALB/c. We also determined if collateral remodeling and angiogenesis after ligation are impaired in BALB/c. Collateral diameters were comparable between strains before ligation. However, diameter increased 54% less in BALB/c by day 21 (Fig. 2A). Reduced outward remodeling of collaterals, and thus cross-sectional lumen area, is consistent with the higher Doppler velocity in the CZ of BALB/c at this time (Fig. 1C). Before ligation, capillary density and capillary-to-muscle fiber ratios were not different between strains in the adductor and were greater, not lesser, in the gastrocnemius of BALB/c (Supplemental Fig. S1). Twenty-one days after ligation, density and ratio had increased in both strains, but to a lesser degree in BALB/c (Fig. 2B and Supplemental Fig. S2, A and B). Ischemia has not been detected in adductor muscle in this ligation model (12, 45). Consistent with this, we found no increase in semimembranosus capillary density or ratio 21 days after ligation (Supplemental Fig. S1, C and D).
These data suggest that, compared with C57BL/6 mice, the drop in perfusion in the CZ and greater reduction in foot perfusion in BALB/c immediately after ligation reflect fewer collaterals, rather than narrower collaterals or fewer capillaries. In addition, the delayed increase in CZ perfusion and reduced recovery of foot perfusion in BALB/c suggest less collateral remodeling and angiogenesis in the ischemic lower leg compared with C57BL/6 mice.
TNF-α in adductor is reduced in BALB/c mice after ligation.
Activation of collateral endothelial cells by increased shear stress causes macrophages to adhere and transmigrate to the perivascular region where their elaboration of growth factors and cytokines is thought to promote collateral remodeling (21, 24). Recently, CD4-positive T cells have been implicated in collateral remodeling (53), specifically the T-helper-1 subtype (Th-1) which produces TNF-α, a cytokine important in arteriogenesis (25). BALB/c have a deficient Th-1 response to parasite infection and antigen challenge (18). Since BALB/c also had impaired collateral remodeling (Fig. 2A), we examined leukocyte density and TNF-α expression in the CZ. CD45-positive (pan-leukocyte marker) cells and CD4-positive T cells increased comparably around collaterals in both strains, suggesting similar recruitment to remodeling collaterals in the BALB/c strain (Fig. 2, C and D, and Supplemental Fig. S2). However, relative to C57BL/6 mice, TNF-α expression was about twofold lower 36 h after ligation in BALB/c adductor (Fig. 2E), in agreement with their deficient Th-1 response (18). Lower TNF-α may contribute to the impaired collateral remodeling in BALB/c mice.
BALB/c mice experience greater ischemia and reduced recovery of hindlimb use.
In BALB/c, the greater drop in perfusion in the CZ and foot immediately after ligation, followed over time by attenuated collateral remodeling, angiogenesis, and recovery of hindlimb perfusion, predicts greater functional impairment. Indeed, the ligated hindlimb of BALB/c had greater tissue damage and loss of use (Fig. 2, F and G). Consistent with this, skeletal muscle fiber size tended to atrophy more by day 21 in BALB/c adductor (−15 ± 8% in BALB/c vs. −5 ± 4% in C57BL/6, n = 10) and gastrocnemius (−61 ± 12% vs. −42 ± 10%, n = 6–7, P = 0.09).
BALB/c mice have fewer collaterals in the hindlimb.
Since baseline collateral diameter and capillary density are similar in both strains, the greater drop in perfusion in the CZ and foot immediately after ligation in BALB/c suggests they have fewer collaterals. In support of this, we detected fewer vessels in the CZ of BALB/c immediately after ligation, by X-ray angiography (performed with contrast material that does not cross capillaries) followed by a Rentrop-like line analysis ("baseline" Fig. 3, A and B). Consistent with this, the number of calf arteries that filled during a constant interval of contrast infusion immediately after ligation (an indicator of collateral conductance) was sevenfold lower in BALB/c (Fig. 3C). BALB/c evidenced a smaller increase in vessel number detected in the CZ at day 7 (Fig. 3B), which is predicted from the smaller number before ligation. Similar results for collateral number and conductance before and 7 days after ligation were obtained by an ROI angiogram analysis (Supplemental Fig. S3, A and B). Both mouse strains showed a decline in vessel number 21 days after ligation (Fig. 3B). This has been ascribed to a renarrowing of a portion of the collateral population that is "permitted" by the persistence of several greatly enlarged collaterals which are sufficient to supply downstream tissue needs at rest (40). To confirm the angiographic data (Fig. 3B), we counted the number of α-SMA+ arterioles (41–100 μm diameter) in the CZ of the semimembranosus and obtained good agreement with the angiographic results (Fig. 3D). These data demonstrate that BALB/c have fewer pre-existing collaterals in hindlimb skeletal muscle. They also confirm the morphometric data demonstrating reduced collateral remodeling in BALB/c (Fig. 2A).
Reduced pre-existing collateral density in BALB/c mice generalizes to other tissues.
To determine if the lower collateral density in skeletal muscle of BALB/c extends to other tissues, we examined small intestine and brain using a fluorescent vascular contrast material that does not cross capillaries. BALB/c had 30% fewer collaterals than C57BL/6 in the jejunum (Fig. 4, A and B). And remarkably, BALB/c were almost devoid of pial collaterals between the MCA and ACA (Fig. 4C). A total of five MCA-ACA collaterals were found in only three of nine BALB/c studied. In contrast, collaterals were abundant in all 10 C57BL/6 examined (175 collaterals total, 15-fold more than in BALB/c) (Fig. 4D). The diameter of MCA-ACA collaterals in BALB/c was also ∼30% smaller (Fig. 4E). Although not quantified, collaterals between the MCA/ACA and posterior cerebral arteries (PCA) were also abundant in C57BL/6 yet virtually absent in BALB/c (Fig. 4C). High-magnification microscopy showed that absence of collaterals in BALB/c was not from failure to detect small ones in the pia or upper cortex. This is consistent with evidence that arteries within the brain parenchyma end in capillaries and do not give rise to collaterals (10). We also did not detect collaterals in vascular casts of BALB/c made with a polyurethane material (28) that filled all vessels including capillaries. This further confirms that intracerebral collaterals are not present in BALB/c mice.
Differences in pial collateral density or diameter among mouse strains have not been reported previously. However, BALB/c have recently been shown to suffer lethal strokes within several days after MCA occlusion, whereas C57BL/6 tolerate the procedure (1, 46). The reason for this difference, which could be from vascular and/or nonvascular factors, was not examined. Therefore, we compared the time required for vascular contrast media infused into the aorta to reach the distal MCA tree after acute occlusion of the main MCA. This time, which is dependent on collaterals connecting the MCA to the ACA and PCA, was about five times longer in BALB/c than C57BL/6 (Fig. 4F). Besides differences in collateral density, the cerebral volume supplied by the MCA (i.e., the size of the MCA "tree") also impacts the area of perfusion deficit and severity of stroke after MCA occlusion (34). Thus, we measured the cortical area overlain by each cerebral artery tree to determine if the MCA distribution was larger in BALB/c. No differences were found between strains (Fig. 4G). Taken together, these data suggest that deficiency of pial collaterals in BALB/c is central to their heightened susceptibility to lethal stroke.
Arterial tree structure differs between BALB/c and C57BL/6 mice.
We noticed during angiographic analyses that the number of distal arterial branches appeared to differ between BALB/c and C57BL/6. Although it is not known if collaterals develop during embryogenesis or after birth, it is possible that their formation is linked to or induced by factors that specify branching characteristics as arterial trees form in the embryo and expand in size postnatally. Therefore, we measured the number of second-order arteries branching laterally from the right profunda and superior epigastric arteries (line 1 and 3, Fig. 5A). BALB/c have fewer branches (white bars, Fig. 5, B and C). Average diameter of the epigastric artery, measured at proximal, middle, and distal points, was similar (C57BL/6 = 177 ± 4 μm, n = 7; BALB/c = 170 ± 4 μm, n = 9). We also measured the number of second-order branches extending from the profunda 7 days after ligation (line 2, Fig. 5A). No changes occurred (Fig. 5B), which strengthens the assumption that arteries detected by angiography in the adductor CZ (Fig. 3B) represent primarily pre-existing collaterals (23).
The higher resolution of fluorescent angiography permitted a more detailed analysis of branching in the pial circulation. Similar to the virtual absence of pial collaterals in BALB/c (above), intra-tree arteriole-to-arteriole anastomoses between adjacent branches within the MCA or ACA trees were abundant in C57BL/6 but absent in BALB/c (Fig. 5D). The reduced second-order branches in skeletal and pial beds, absence of anastomoses, and additional differences in the pia [i.e., branching angles and numbers of arterioles at more distal branch orders (see legend of Supplemental Fig. S3)] demonstrate that the arterial tree structure in skeletal muscle and pia differs between BALB/c and C57BL/6.
VEGF-A expression is reduced in BALB/c mice.
Recent studies are beginning to identify factors, specifically VEGF-A isoforms, which are important in specification of vascular branching pattern at different stages of embryogenesis (8, 26, 42, 56). It is possible that formation of collaterals and thus collateral density may also be determined during this specification and be dependent on VEGF-A. VEGF-A is paramount in capillary sprouting during ischemia (16), which was strongly reduced in BALB/c gastrocnemius. Thus, we investigated whether BALB/c are deficient in VEGF-A expression. Expression of VEGF-A isoforms was determined in the tissue samples of adductor CZ and gastrocnemius before and 36 h after femoral ligation using quantitative real-time PCR. After ligation, VEGF-A in the adductor CZ did not increase in either strain, which is in agreement with absence of ischemia (12) and angiogenesis therein (Supplemental Fig. S1B). VEGF-A in gastrocnemius before ligation trended lower in BALB/C, and VEGF-A-164 and −188 increased 3- and 18-fold more in ischemic gastrocnemius of C57BL/6 than BALB/c (Fig. 6). These data suggest that impaired VEGF-A expression may underlie the deficient angiogenesis and reduced collateral density in BALB/c mice.
Gene polymorphisms in BALB/c mice may underlie their low expression of VEGF-A.
Expression levels of VEGF-A and TNF-α were markedly lower in gastrocnemius of BALB/c mice after femoral artery ligation, even though ischemia was greater than in C57BL/6. Basal expression of VEGF-A also trended lower in BALB/c. VEGF-A is capable of increasing tissue levels of TNF-α directly (35, 63) and also indirectly through leukocyte recruitment. Moreover, TNF-α is important in collateral remodeling (25). Recent evidence implicates VEGF-A in embryonic vascular branching morphogenesis (8, 26, 42, 43). These findings suggest that, compared with C57BL/6 mice, BALB/c may have reduced VEGF-A transcription (and/or altered splicing and transcript stability) to contribute to their altered branching pattern and lower collateral density, reduced TNF-α expression and collateral remodeling, and impaired angiogenesis. To begin to explore this possibility, we conducted a bioinformatic analysis to test for presence of eQTL associated with variation in mRNA abundance for VEGF-A. We also mapped eQTL for genes "upstream" of VEGF-A that regulate its expression or that reside within its downstream signaling pathway. We used a hippocampal mRNA microarray dataset that was generated from a recombinant inbred set of 13 genetically distinct strains derived from the F2 cross of C57BL/6 and BALB/c mice (CXB, 7, 14, 61).
Interval mapping (Fig. 7, Supplemental Table S1) identified a putative eQTL (LOD ≥3, unless stated otherwise) for VEGF-A expression within ∼10 cM ("cis") (13) of the Vegfa gene on chromosome 17 (MMU17, microarray probe sets did not distinguish among VEGF-A isoforms). This putative cis-acting eQTL in the BALB/c allele is associated with reduced VEGF-A expression relative to C57BL/6. This intriguing finding suggests that BALB/c may have a polymorphism in or near Vegfa that results in the reduced expression that we detected. eQTL for HIF-1α (LOD 2.8) and AP-2, key transcription factors for VEGF-A (39), were also detected that overlap with the MMU17 VEGF-A cis-eQTL (Fig. 7, Supplemental Table S1) and associate with reduced expression in BALB/c.
An eQTL for the VEGF-A-activating morphogen, sonic hedgehog (Shh), was identified that associates with increased expression in BALB/c and overlaps the MMU17 Vegfa eQTL (Fig. 7, Supplemental Table S1). Shh induces, through downstream VEGF-A signaling, induction of Ephrin-B2, which specifies the arterial endothelial cell phenotype during embryonic vascular morphogenesis (30). Thus, this association of increased Shh expression in BALB/c with the cis-acting inhibitory eQTL at MMU17 Vegfa may represent a compensatory response. Interval mapping of Ephrin-B2 identified an eQTL that overlaps with the MMU17 Vegfa eQTL and is associated with lower expression in BALB/c, which may result from reduced expression of VEGF-A. SHP-1 and SHP-2 are cytoplasmic protein tyrosine phosphatases that negatively modulate VEGF-A signaling by dephosphorylating VEGF receptor-2 (Flk-1) (27), which is the receptor that mediates VEGF-A-induced angiogenesis (51). Recently, knockdown of SHP-1 by siRNA was shown to augment angiogenesis in ischemic hindlimb (55). We therefore investigated SHP-1 and SHP-2. An eQTL that overlaps the MMU17 Vegfa eQTL is present for SHP-2 but not SHP-1 and is associated with increased expression in BALB/c (Fig. 7, Supplemental Table S1). Thus besides reduced VEGF expression, BALB/c may also have impaired Flk-1 signaling. The angiogenic factor FGF-2, which is known to induce expression of VEGF-A (48), was also examined. Interestingly, an eQTL (LOD 2.8) that overlaps the MMU17 Vegfa eQTL is present for FGF-2 and is associated with increased expression in BALB/c, which could be compensatory for low VEGF-A expression. These colocalized linkages for expression of multiple genes that either drive or are driven by VEGF-A suggest that regulation of VEGF-A expression and its downstream signaling may differ systematically between the C57BL/6 and BALB/c strains and may be caused in part by a single cis-acting QTL at the Vegfa locus itself.
Collateral density in normal tissues and collateral growth in ischemic disease are known to vary widely among species and human subjects (62). Genetic variation has long been suspected to contribute to these differences, but this possibility has not been examined. Identification of differences among inbred mouse strains in developmental, physiological, and pathological processes provides a first step toward investigating genetic contributions. To this end, we describe pronounced differences in BALB/c vs. C57BL/6 mice in angiogenesis, collateral density and remodeling, and VEGF-A expression. We also report preliminary yet provocative data showing evidence for a putative eQTL near the Vegfa gene that may account for the variation in mRNA abundance for VEGF-A (in cis) and for several additional genes (in trans) both upstream and downstream of VEGF-A signaling in the CXB recombinant inbred strain set.
BALB/c mice evidenced reduced angiogenesis and collateral growth in the hindlimb after femoral artery ligation. This was associated with lower expression of VEGF-A and TNF-α, cytokines important in capillary sprouting and collateral remodeling in ischemia, respectively. Compared with C57BL/6 mice, BALB/c also had significantly lower density of pre-existing collaterals in skeletal muscle and intestine and, most notably, almost complete absence of collaterals in the pial circulation. The lower density in skeletal muscle was recently reported (22), but extension to other tissues or underlying mechanisms was not examined. We found that the deficiency in collateral density in BALB/c mice resulted in greater ischemic damage in skeletal muscle after femoral artery ligation and impaired perfusion of the cortex after MCA occlusion. This latter finding provides an attractive hypothesis to explain the recent observation that BALB/c suffer lethal stroke within 3 days after MCA occlusion, whereas C57BL/6 mice are spared (1, 46). However, results in a recent study (36) appear at variance with our hypothesis that the difference in size of cerebral infarction between the strains resides in their marked difference in collateral abundance. Majid et al. (36) did, in fact, observe a threefold larger infarct volume in BALB/c than C57BL/6 mice after MCA occlusion, which is in agreement with our results and those of others (1, 46). However, they measured a smaller difference in perfusion between the strains than predicted from our results. This likely reflects the fact that perfusion was measured in a restricted region with a Doppler flow probe that overlaid a small craniotomy. In addition, this arrangement simultaneously sampled dural flow, which is not supplied by the MCA.
Substantial heterogeneity has been reported in in vitro assays of hypoxic stimulation of VEGF-A expression by monocytes from patients with coronary artery disease (47). Likewise, significant variation in VEGF expression in human myoblasts has been linked to haplotypes in the VEGF-A gene (41). Other studies also suggest genetic variability in factors controlling VEGF expression (29, 54). Given these findings and the differences in VEGF-A expression that we observed, we conducted eQTL mapping of VEGF-A and associated genes using a database generated for the CXB recombinant inbred set of mice. Evidence for a cis-eQTL near the Vegfa locus, associated with low VEGF-A expression in BALB/c, may represent polymorphisms in the Vegfa promoter, enhancer, or other regulatory elements that result in the reduced VEGF-A expression we observed in BALB/c relative to C57BL/6. We also identified eQTL for the key VEGF-A transcription factors, HIF-1α and AP-2 (39, 49), that overlap the MMU17 cis-Vegfa eQTL. These eQTL are associated with reduced expression in BALB/c. These findings suggest possible cis- and trans-acting mechanisms that could underlie reduced VEGF-A expression in BALB/c mice (Fig. 8).
We also found eQTL overlapping with the MMU17 cis-acting Vegfa eQTL for ephrin-B2 and angiopoietin-1, which are in VEGF-A's downstream signaling pathway and are important for differentiation of endothelial cell, vascular growth, and remodeling (20, 59), that were associated with reduced expression in BALB/c, relative to C57BL/6; moreover, an MMU17-overlapping eQTL associated with increased expression of the phosphatase, SHP-2, in BALB/c was also identified, which could further impair VEGF-A downstream signaling through decreased phosphorylation of VEGF receptor 2 (Flk-1) (27). These findings suggest possible deficits in factors within VEGF-A's downstream signaling pathways that could contribute to the differences in pre-existing vascular morphology and adaptive angiogenesis and collateral growth in ischemia exhibited by BALB/c mice (Fig. 8).
The power of eQTL mapping in the small (13-line) CXB recombinant inbred line (RIL) set is very limited. Because of this, we have only focused our analysis on the region of MMU17 where Vegfa is harbored to examine the hypothesis, analyzed by a single statistical test, that eQTL exist at this locus affecting VEGF-A expression. After finding evidence for a potential cis-eQTL associated with low VEGF-A expression in BALB/c, we then tested a second specific hypothesis, i.e., that this eQTL also associates with variation in mRNA abundance for genes involved in VEGF-A signaling. Our results showing a putative cis-eQTL for Vegfa that also has trans-acting effects on several transcripts related to VEGF-A signaling provide provocative hypotheses for future study. In particular, the possibility that one or more eQTL may underlie the low VEGF-A expression in BALB/c mice needs to be confirmed through sequencing and subsequent studies. Furthermore, studies are underway to measure collateral density in the CXB RIL, to test the hypothesis that a QTL for collateral density overlaps the MMU17 Vegfa eQTL. Additional power could be achieved by employing the recombinant inbred cross strategy for CXB as proposed by Zou et al. (64) or by using a large F2 cross between C57BL/6 and BALB/c for QTL and eQTL mapping. Future studies also need to evaluate eQTL using mRNA derived from vascular sources during formation of collaterals in normal tissues and collateral remodeling in ischemia and to confirm expression differences with real-time RT-PCR. Finally, sequencing of VEGF-A from BALB/c and comparison to the existing C57BL/6 genomic sequence may identify polymorphisms with potential effects on transcription.
The above mapping differences in BALB/c and C57BL/6 mice lend support to evidence from previous studies suggesting a role for VEGF-A signaling in collateral growth in ischemia (6, 31, 57). Moreover, they provide insights regarding genetic factors that may underlie the altered arterial branching pattern and large deficit, especially in the pial circulation, that we observed in density of pre-existing collaterals in multiple tissues of BALB/c mice. This association suggests that determinants of branching pattern or outgrowth of the arterial tree during development differ between these two strains and may underlie differences in collateral formation. Little is known about such determinants; however, VEGF-A has recently been implicated in branching pattern (8, 26, 42, 43, 56). While only a single VEGF-A isoform is required for embryonic capillary formation (32, 33, 38), evidence suggests that the final vessel architecture is specified by gradients established by expression of VEGF-A isoforms (42). We hypothesize that collaterals form during the specification of arterial tree structure and that altered VEGF-A expression/gradients in BALB/c mice result in reduced formation of collaterals. Other studies underway within our group support this possibility. We have found that pial collaterals form during the late embryonic-to-early postnatal period and reach the adult density by 3 wk after birth in C57BL/6 mice (3). By comparison, BALB/c have formed 50% fewer collaterals by birth, and their diameters are smaller. Most interestingly, these collaterals are lost by 3 wk after birth, suggesting they may fail to mature. In addition, we have direct support that VEGF-A is important in collateral formation. Mice with targeted mutations of the VEGF-A gene resulting in either increased (37) or decreased (11) VEGF-A expression have marked increases and decreases, respectively, in collateral density in multiple tissues (9, 15). Consistent with this finding, the mutants also have smaller vs. greater perfusion deficits, respectively, immediately after femoral artery ligation and better vs. worse recovery of perfusion and hindlimb use thereafter.
While nothing is known regarding how collaterals form in normal tissues, our above (3, 9, 15) and present findings suggest a model for perinatal collateral formation (Fig. 8). Accordingly, we postulate that insufficient VEGF-A expression in BALB/c compared with C57BL/6 mice, arising from the above gene polymorphisms in HIF-1α and AP-2, together with reduced downstream induction by VEGF-A of Eprhin-B2 and Ang-1, contributes to reduced formation of arteriole-to-arteriole cross-connections (collaterals) between adjacent arterial trees. Interestingly, we also identified a single eQTL for Ang-2 (MMU7, 132–140 Mb, LOD 4.4, data not shown) associated with increased expression in BALB/c. It is well known that newly formed vessels fail to mature and are pruned away in the context of low VEGF-A, low Ang-1, and high Ang-2 activity (20). This is consistent with our observation that the reduced number of collaterals that do form in the BALB/c embryonic pial circulation fail to mature and are pruned away by P21 (3). It is important to note that hemodynamic factors and other genes are undoubtedly also involved in collateral formation and maturation.
The striking difference in collateral density between BALB/c and C57BL/6 mice, together with the availability of the CXB RIL strain set, underscores their potential as a model to uncover the signaling mechanisms that direct collateral development in normal tissues and collateral remodeling in ischemia. Determination of these mechanisms will likely provide insight into the genetic mechanisms underlying the marked variability in collateral density and remodeling among species and humans. Such information could lead to novel strategies to induce formation of new collaterals and enhance remodeling of existing collaterals in patients with ischemic heart disease, stroke, and peripheral vascular disease.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-62584 (J. E. Faber), F32-HL-080847 (D. Chalothorn), and T32-HL-069768 (J. A. Clayton).
We thank Dr. Victoria Bautch for critical comments on the manuscript and Kirk McNaughton and Carolyn Suitt for histology.
↵1 The online version of this article contains supplemental material.
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: J. E. Faber, Dept. of Cell and Molecular Physiology, 103 Mason Farm Rd., 6309 MBRB, CB 7545, Univ. of North Carolina, Chapel Hill, NC 27599-7545 (e-mail: jefaber{at}med.unc.edu).
- Copyright © 2007 the American Physiological Society


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Thanks in part to inexpensive reality shows, new television series are having a hard time making it past a single season. Taking the time to sit down and really get involved with new characters seems futile, when the likely end result will be cancellation and disappointment. It's amazing when shows that began ten to fifteen years ago - and ran a commendable five to seven seasons - come out on DVD because it's a reminder of what might be lost on today's audience. A great example of that is recently released "Spin City: The Complete Fifth Season." As I took another look at the series on DVD, I can't help but wish for a return to the '90's, when television was, well, good.
"Spin City" was a sitcom that took place in city hall. While the environment would seem more suited to a cop drama, "Spin" managed to get a remarkable amount of laughs thanks to solid writing and a terrific cast. "Spin City" isn't really a show about politics, instead it was a platform to collect a group of individuals that were often at odds, creating funny, standard sitcom situations. What made it work as well as it did was the consistent writing and witty performances.
When "Spin City" first aired, Michael J. Fox did a fantastic job leading the show as deputy mayor, Mike Flaherty. His job - as well as the rest of the staff's - was to keep the Mayor (Barry Bostwick) from drawing any negative attention to his somewhat dim-witted behavior. The series did a great job focusing on a different storyline each week, giving the staff something to take care of, and often Flaherty to help save the day. While not as memorable as same-era sitcom, "News Radio," there's a similarity in the way the group dynamics play off one another, creating a handful of humorous situations from week to week.
By the 4th season, Fox decided it was time to leave the series, and the creators decided to carry on with a new lead character. Interestingly enough, the replacement for Fox was Charlie Sheen (who was recently replaced after he left the series, "Two and a Half Men.") Ratings suffered in the fifth season without Fox's character, and Sheen - especially in retrospect - seems like too significant a change in comedic tone at the core of the show. Staffers that continued on to season five included press secretary Paul (Richard Kind), head of minority affairs Carter (Michael Boatman), chief of staff Stuart (Alan Ruck), and season four addition, campaign manager Caitlin (Heather Locklear). Charlie Sheen played the new deputy mayor, Charlie Crawford who had a different approach to dealing with the Mayor and office staff. Unlike Flaherty, Crawford is a bit egotistical, which doesn't always go over well with his new co-workers, mainly Caitlin. Still, despite some character differences, the show basically stays the same course.
The thirty minute episodes focus on things like Caitlin trying to improve her personality when a blind man finds her unappealing ("Blind Faith"), Stuart, Carter and Paul all compete to get to go on a trip with the mayor to New Orleans ("Lost and Found"), the Mayor's ego is deflated when Charlie plays him in a game of racquetball and wins ("All the Wrong Moves"), when the Mayor's ratings go down, Caitlin hires an image consultant that leads to interesting results ("The Image Maker"). While there's nothing necessarily new to the plots, the actors do help carry the series with the same energy and delivery as previous seasons. Sheen wasn't a perfect choice to replace Fox and the shift in tone leads to a series that isn't as satisfying or quite the same. However, it's certainly not bad and still delivers a good deal of laughs.
With no extra features, "Spin City: The Complete Fifth Season" is recommended for fans of the series who want to add to their collection. For those unfamiliar with the series, I recommend starting with the earlier season and working your way through them to the fifth. It's a series that's certainly worth a look for the memorable performances, humorous situations, and if anything else, it's a reminder of what television used to be: consistent entertainment.
Season 5:
5-01 18/Oct/00 Hello Charlie
5-02 25/Oct/00 Smile
5-03 01/Nov/00 The Spanish Prisoner
5-04 08/Nov/00 The Bone Collectors
5-05 15/Nov/00 Blind Faith
5-06 22/Nov/00 Balloons over Broadway
5-07 29/Nov/00 Lost and Found
5-08 06/Dec/00 All the Wrong Moves
5-09 13/Dec/00 The Burgers of Wrath
5-10 20/Dec/00 Toy Story
5-11 10/Jan/01 The Perfect Dorm
5-12 17/Jan/01 Hey Judith
5-13 24/Jan/01 The Gambler
5-14 07/Feb/01 In the Company of Dudes
5-15 14/Feb/01 The Image Maker
5-16 21/Feb/01 Trainstopping
5-17 28/Feb/01 Rain on My Charades
5-18 25/Apr/01 You've Got Male
5-19 02/May/01 Minor League
5-20 09/May/01 Science Friction
5-21 16/May/01 Brotherly Love
5-22 23/May/01 A Shot in the Dark (part 1)
5-23 23/May/01 A Shot in the Dark (part 2)
The DVD
VIDEO: Shout Factory gives the show a very nice presentation. Offered in 1.33:1 full-frame, the series looks bright and crisp. While not crystal clear, the series looks about broadcast level. Colors look warm and crisp, with no smearing or other faults.
SOUND: Clean, well-recorded stereo soundtrack, with clear dialogue.
There are no extra features.
Final Thoughts: "Spin City" starts to lose altitude in the fifth season with the departure of Fox, but so-so "Spin City" is still better than most of what's on TV today. Recommended, although those new to the series should certainly start off with the first season.

subset: commoncrawl


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Here are some places in the city to indulge in an array of fall colors.
Occupying the top nine floors and the first three levels of the 53-story Midtown Tower, The Ritz-Carlton, Tokyo transfixes visitors with a world-class rejuvenating spa, acclaimed restaurants and a skyline view from this luxury hotel's exquisite guest rooms. Also from this city-center location, guests can enjoy the vibrant night life and centuries-old attractions of the Roppongi District, Tokyo's cultural, diplomatic and entertainment heart.
The Ritz-Carlton, Tokyo is a Tier 5 property in The Ritz-Carlton Rewards.
Here are some places in the city to indulge in an array of fall colors.
When you have more time in the city, you can make more memories. The Reconnect® package affords you enough time to enjoy everything the city has to offer. How will you Reconnect?
We are excited with the arrival of beautiful autumn, when leaves are changing, streets and gardens are bursting with pretty colors, and minds are filled with warm autumn memories.Learn More
Experience our club lounge featuring four distinctly unique spaces and exclusive privileges.
Escape to a Tokyo spa hotel that inspires renewal. Providing spectacular panoramic views from its perch on the 46th floor, The Ritz-Carlton Spa & Fitness offers guests the ultimate pampering, rejuvenation and restorative experience in a tranquil sanctuary.
Explore Tokyo - the modern capital that features cosmopolitan nightlife, incredible entertainment and modern Japanese culture. Immerse in local culture and partake in hotel's customized art of Japanese arts of the crafts.
Explore the area by connecting with us on foursquare, where you can discover others' favorites and share your own.


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Friday, April 26, 2013
Jerry Brewer: If the Seahawks weren't so successfully weird, you would overreact now. They took backup running back Christine Michaels, above, a possible third-stringer, in the second round of the NFL draft Friday.
Photography home | Most viewed photos | Purchase photos | Video home
© 2013 The Seattle Times Company


text: I was walking with some friends through the food court at the mall one Saturday. The mall was crowded and the food court was even more crowded. I was trying to look like I didn't notice these cute guys we were walking by. When all of a sudden I tripped and fell flat on my face. It was so embarrassing.


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Separate names with a comma.
Discussion in '1996 - 2004 SN95 Mustang -General/Talk-' started by spdsht, Jul 11, 2013.
looked into a wrap for my car they wanted 3000-3500 haha I was like oh that's like a new paint job.
Wow that's extremely high, I've seen them for $1,500
-Nick C.
If that's what it was I would get it done for suuuuuure but nope. I was kinda choked when they told me the price, I expected a lot less, and I am not sure how I feel about the door james, under hood and trunk and such not matching the exterior
Actually, blue door jams look pretty sweet on a black car sometimes...especially if you don't want to pay to have a full paint job.
I saw a new black 5.0 that was kona blue and the guy wrapped it.
Nick C.
tbh I was thinking flat blue would look nice there are some on youtube.. what color black, like shiny or flat?... I think id be nervous of it tearing or something making it look really bad compared to a paint scratch... ideas on that?
I've actually seen some sweet flat blue cars as well. Vossen did a video with the most gorgeous flat blue Tesla Model S, I've ever seen. Totally sold me on the color:
View: http://www.youtube.com/watch?v=KtKbGI92ipY
So beautiful...plus I love where they filmed it, makes me laugh every time
Nick C.
Absolutely gorgeous car, and it's American, so import bashing people can't hate on it.
yah I love the color just would probably need the door jams painted black to look good since its a diff shade of blue may look off... is the wrap durable? would hate to have it rip or tear or get scuffed up and look like
It's pretty durable from what I hear, it's becoming very popular as well.
It doesn't even damage the paint it's applied over typically, pretty interesting stuff IMO.
Plus, I'm sure a reputable shop would back it with some kind of warranty
I wonder if your paint needs to be perfect underneath? would rock chips and such show imperfections in the wrap
Probably not too bad, unless you have massive dents
Just watched the Tesla video. Wow....flat blue.....who new that looked so good. Especially with the dark colored wheels and tinted window.
I think vinyl wraps are a good idea and fairly durable. I saw a guy who bought a brand new Camaro ss in black and had the whole thing wrapped in flat black. Looked sweet and he said he drove the car everywhere. When he wants to change the color up, peel and re-apply.
That's the coolest thing about it, plus it protects the paint underneath from most stone chips, bird poo, etc...
I'm usually a black car only guy, but I could see myself putting a wrap on a car someday...well not me personally, I'd pay to get it done lol
-Nick C.
yah except they cost about 3000 or more hah.. that's pricey to just peel off and change... and what if the wrap gets damaged, if you get that panel repaired will it look off .. since the rest will have been weather and sun faded im sure
They are surprisingly weather resistant, and usually cheaper than $2,000
Depends where you live I suppose
Nick C.
Honestly, if you do that you're gonna look like one of those import guys driving their Honda's around with that "work in progress" look that they think is so cool. Might as well hang a stuffed panda from your rear bumper too...
Vinyl wrapping looks really good when it's done right. Problem is, getting it done right is much more expensive and labor intensive.
Nick C.


text:
What I have been trying to do in these three lectures is to focus attention upon one or two points at which civilization itself seems to be threatened by contemporary tendencies—tendencies not in themselves deleterious, tendencies which if restrained within certain limits are actually civilizing tendencies, but which are dangerous if they are carried too far.
First, civilization depends upon the recognition and the cultivation of excellencies and superiorities; the maintenance of a civilized society, therefore, would be made impossible by a thorough-going egalitarianism. Competition in excellence is a civilizing force, and you do not discredit it by calling it a rat-race.
Second, civilization depends upon the maintenance of law and order, and law and order can't be maintained, in an imperfect world, without resort to force. Humanity, in the sense of humaneness, is not self-sustaining. You do not discredit force by calling it brute force.
Finally, civilized society exists for the individual—we can't remind ourselves of that too often in an age when the state has grown top heavy, and seems to crush all individual effort, all individual character, to leave us little room to fulfill ourselves: no wonder there exists today an almost desperate craving for self-expression. But society is pluralistic, and each individual must respect the individuality of his fellow citizens; he must exercise self-restraint, out of respect not only for the rights but for the feelings of his fellow men; self-expression is not self-validating, and affords no sovereign plea in its own defense when it collides with the common feelings and susceptibilities of mankind.
(John Sparrow, Too Much of a Good Thing [Chicago and London: The University of Chicago Press, 1977], 90-2)


text:
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wikiHow to Make a Tidal Wave Cocktail
Take a sip of this drink and you'll feel like your surfing a huge wave without being in the ocean. Try this recipe on how to make a tidal wave cocktail.
- 0.5 ounce (15 ml) of vodka
- 0.5 ounce (15 ml) of tequila
- 2 ounces (50 ml) of pineapple juice
- 1 ounce (30 ml) of orange juice
- 1 teaspoon of grenadine
- 1 cup of crushed ice
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7 Comments
really love that light color combination :))
Nice, it looks a bit too transparent though imo .. but love your art mate! +1
Thanks! I'm kinda new at this so I wasn't sure if I could go that light or not. I'll keep that in mind for next time :)
dandingeroz Artist from Philippines
cool +1
cool!sure i vote for this


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Union Historical Society will offer its annual narrated bus tour of early historic sites during the town's Founders Day weekend. Tours depart from the Old Town House on Town House Road, just off Union Common, at 5:30 p.m. on Friday July 18 and at 9:30 a.m. on Saturday July 19. All the sites visited during the 2-hour tour are related to the settlers mentioned in Come Spring, Ben Ames Williams' 1940 historic novel, which in turn is based on information in John Langdon Sibley's 1851 History of the Town of Union. Four of the sites are located on private property and are not open to the public at other times. Tickets are priced at $12 for the Friday tour and $25 for the Saturday tour, which is in an air-conditioned coach. To purchase tickets, call Union Historical Society at 785-5444 and leave a message, or come to the Old Town House at least 30 minutes before the tours depart.
Come Spring Bus Tours of Union's historic sites
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text:
- The Bella Twins are scheduled to make their first post-WWE appearance at an autograph signing on Saturday, May 19th at the Frank & Son Collectible Show in Industry, California. Autographs and photos will be available for $30.
Maybe they part ways with WWE for their own good and for WWE's, but I miss them already…
I'm Still Not Conviced That They not still with the WWE ….. in some way
It sucks that they are gone. They are one of the few divas that I actually liked watching.


text:
Don't subsidize junk mail
The "Forever Stamp" may not last forever if the Postal Service doesn't get a chance to charge EVEN MORE for direct mail. If they continue to offer DISCOUNTS to ostensibly increase volume (NOT PROFITS) as was reported in the U-T ("USPS plays favorites on advertising," Editorial, Aug. 28), then the Postal Service's very existence may be in jeopardy!
Congress has not allowed increases in direct-mail rates as it should, but it should at least prohibit any rate cuts for such a losing program, forcing USPS subsidy costs onto first-class. Junk-mail users should be forced to pay their fair share and NEVER get rate cuts until the USPS is solvent! – John de Beck, Bay Park
Letters and commentary policy
The U-T welcomes and encourages community dialogue on important public matters. Please visit this page for more details on our letters and commentaries policy.
- E-mail firstname.lastname@example.com
- Mail: Andrew Kleske, Reader Outreach Editor
San Diego Union-Tribune
P.O. Box 120191
San Diego, CA 92112-0191.
Support for Prop. 35
Thanks to Greg Moran for bringing to readers' attention the alarming extent of the crime of child sex trafficking in San Diego ("Child sex trafficking, pornography cases up 545%," Courts, utsandiego.com, Aug. 23). This is a truly heinous crime, and, though its pervasiveness within our own fair city is only lately coming into focus, it occurs domestically and exploits girls often as young as 12 and from myriad backgrounds.
That is why California needs to vote Yes on Prop 35 this November.
Moran's report observed the role of organized gangs in this modern form of slavery. Prop. 35 will make such money-motivated criminals reconsider the lucrativeness of this crime, deterring them with increased prison terms for convicted traffickers. It will increase fines against traffickers and direct that money to victims' services. Prop 35 will also require convicted sex traffickers to register as sex offenders, and, recognizing that the Internet has become a nearly ubiquitous element across cases of child sex trafficking, it will require registered offenders to disclose their Internet accounts.
I urge voters to take a stand against the sexual exploitation of our children by voting Yes on Prop 35 this November. – Henry Fung, San Diego
Is city getting what it paid for?
I've been noticing a disturbing trend in San Diego: bad purchasing decisions for the city. If you've ridden a bus lately, you've probably noticed you can't understand a thing the driver says on the new public address systems. It's too loud and it's completely garbled. When we ride we make a game of trying to understand it, but it usually sounds like nonsense.
And have you noticed the resurfacing of the streets in San Diego? It's a shoddy job, worst I've ever seen. It's more like painting the streets black and hoping no one will notice. The street where I live still had not had the lines repainted after more than two months. And you can scuff up the half-inch of tar with your shoe, so you can imagine what cars are doing to it.
This is the kind of thing that impacts us as San Diegans every day. Who's making these bad decisions? – Tom Ross, Hillcrest
Water worries
Your article "Southern California escapes effects of drought – for now" (Aug. 26) prompts the following comments:
While San Diego may not be experiencing drought conditions at present, this is a desert area and we don't have enough water to support our population. Yet we seem to follow what appears to be the typical American response, namely we wait until the eleventh hour and then panic and suggest water rationing of some kind.
Here are some of the things I think we should be doing:
(1) Consider a building moratorium. How in good faith can we allow the construction of additional living units when we really don't have a safe water supply for existing units?
(2) Any new construction permitted should be required to capture all but toilet water to be used to irrigate the grounds.
(3) While they are expensive to build, we should bite the bullet and take advantage of the huge supply of water existing along our shore and build desalination plants, and prohibit the lawsuits and red tape that have delayed the one in the works by almost 10 years and greatly increased costs.
On another subject, namely the July Fourth fireworks display – or lack thereof – it seems that the city signed a very poorly worded contract that allows the contractor to now offer a credit for next year that appears to be for a sum considerably less than the city paid. If this is true, who is responsible? Do we not have competent people handling such matters, and if so, should not someone be fired for incompetence? Why has nothing been published about this? – Warren Ralph, San Diego
Audit audacity
It's clear why California is hemorrhaging cash, after the state attorney general claims it will take five MORE people to audit a money stream of $800,000 ("Charity bingo game's number may be up," Aug. 27). You have to be kidding, right? No business with $800,000 gross sales in 18 months has that many full-time auditors. There are nowhere near that many auditors per $100,000 of expenses for the state, like Medi-Cal, welfare or education. ONE person could audit each of the eight nonprofits that offer venues for one month and still take their usual civil service four months off every year. Yes, it will be difficult for them to work more than six months a year at a state government position, but we all have to deal with today's hard economic times.
If the state's attorney general truly thinks it needs five MORE people to audit this insignificant cash stream, then the entire attorney general's office should be replaced with competent people who know how to deal with money in the real world. The era of government empire-building is OVER! – David P. Stroup, San Diego
Bear was in his element
Richard White's death is tragic and I offer my deepest condolences to his family ("Victim of bear attack loved to hike solo," Aug. 27). I don't understand why the bear had to die, too. When we go hiking in places like this, we are on their turf. They are wild animals, and maybe the bear unfortunately felt threatened. We cannot hold a wild animal accountable for protecting itself in its own environment.
Killing the bear doesn't bring White back. If we're going to kill a bear every time one attacks someone, we might as well just kill them all now. There have been similar tragedies with other people hiking alone, which would indicate it's probably wisest to hike with others. – Melanie Ross, North Park
Mount Soledad cross debate
Too bad Mary Sanchez (letters, Aug. 27, about the Mount Soledad cross) doesn't understand the guiding principles of our republic. That no majority shall ever hold sway over the minority in this nation is what makes us great. Weekly votes as to whether the Mount Soledad cross should stay or go – with every single San Diegan voting to let it stand – would do nothing to the change the fact that a religious symbol on public land is still unconstitutional.
As it should be. – Ellen Brown, San Carlos
[The letter's headline read, "Give the public what it wants, let the cross stand."] As long as they are Christian and not Jews, Muslims, Buddhist, Hindus, Wicks, Atheists, Agnostics, etc. After all, there are only Christians who die in foxholes. – Sam Warren, San Diego


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In Short
DIY — short for Do It Yourself — is a weekly column by Ally Kazmucha that focuses on cool and useful projects you can do with — and to! — your iPhone, iPad, and Mac. Whether it's field stripping and fixing your out-of-warranty devices or creating something amazing, DIY aims to bring your ninja skills to the next level.


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The first thing I noticed about Adou Katavasi is the opening riff in track one. It was powerful and ambient, setting the audience on edge. It is clear from the beginning of this that the artists appreciate the genre enough to appease its beauty unto the listener. Yet, at the same time it is thrashy and heavy, the perfect mix of hatred and avarice that makes any listener delve right in to the ever-changing riffs.
The second track in this demo starts off with a different sort of energy than its previous track, which is desirable in my opinion unless one is going for a conceptual type of album in the constant chase of defining mood with the discordant strings and cacophonous harmonies. Soft and fast. Soft and fast. In this design we find order in track two. Its abrupt end made me happy, showing me that the artists weren't afraid to cut the beauty off to the audience, showing that they are indeed in control of their artwork.
Track 3 is thrashy and hateful. Powerful and war-mongering. It had me head banging about 20 seconds into the track and kept it steady while the majority of the track was defined mostly by the drums as well as palm-muting of the guitars. Short and sweet, or in this case, anti-human.
Under the Wings of Thanatos is an interesting song in the simplicity of the guitar riffs mixing with the complexity of the rest of the instruments. The lead vocalist's bellows move along with the beats and drives the song forth with strength anger. It is a good outro to this demo album from Erevos, especially considering it gives the audience something to reminisce on after the album draws to a close. It is catchy, disgruntled, and at the same time, completely uncaring of what the viewer would perceive.
To wrap everything up, I would say Erevos, at least judging from this demo album, reminds me of how the black metal scene developed in Sweden, versus Norway. The style is similar to both Marduk as well as Thy Primordial, maybe mixed in with a little of their own way of doing things, and leaves the audience, at least those who can sit back and truly reflect on the music they're listening to, feeling satiated, like a good workout or smashing some asshole's face after they said a rude comment. I give this album 85 out of 100, in the hopes that they will elaborate on what they have going on here. I'd recommend anyone interested in the underground scene to check this band out!
Well, it has been some time since I have written on a 'single' release... especially on one whose length is around sixteen minutes. Normally such are spread as a demo, sometimes even as an ep; this band from Greece thought different, though. (Yeah, it is a pretty lame introduction, but I could not think of something better)
Erevos play black metal with symphonic as well as thrash facets and the four compositions on Adou Katavasi are well crafted, catchy and interesting written. To make one aspect clear: the keyboards do not drown the rest of the instrument or vocals respectively, but appear in the back as some kind of texture or facet in order to give the music some additional volume. This role works fine with the concept of the Greek band and helps them to create a more fascinating atmosphere. In terms of the arrangements, then guitars and synths form some kind of unity and there are moments in which these would play the same motives parallel to each other; the keyboards do not always appear though or merely orient their play on the guitar lines, so the harmony is not a permanent facet of the music. Such minimalism can backfire as the compositions tend to be more bland and get more predictable through this. Yet, Erevos varied the concept enough to avoid this pitfall.
The music reminds on several bands: Ork, Riddle of Meander and a bit on Dawn from Sweden and also Dimmu Borgir. This is not 'pure' black metal, as influences from other genres have their place in the oeuvre of the Greeks; thrash for instance and also glimpses from the death metal are not far off. Especially the faster segments of the music make the music interesting, as they come with a lot of drive and neat arrangements; Those who decide about fate for instance. The songs are occasionally quite straight-forward and do not come with too much progressiveness.
Even though a lot can be praised on this single, sixteen minutes in length!, there are also some aspects that need to be criticized. First of all, is the song-writing not entirely convincing. Occasionally, the band drifts off into too much repetition, too plain segments or even too many vocals. Yes, the instruments have the room to unfold their potential, but I would like to have an ounce more, please. To get really nit-picky, the production is not perfect, even though it was well executed; the last track comes with vocals which are perceived a bit off the limit and not coherent with the overall sound. As this composition is the only one where this strange aspect can be found, I am inclined to say that it is an exception... a small (negligible) flaw.
Final bits and bytes
The quality of the demo is good and the powerful production played an important role to create this impression. Of course there is room left for improvement still, but it is possibly to really enjoy this piece of art. Some effort was spent on the creation of this piece of music and the listener will appreciate this with every further spin. The songs are interesting and offer the listener enough to motive this person to return to it again.
Even though I am able to appreciate the effort of Erevos, I am inclined to say that their art is not something I would be able to enjoy over a longer period of time. Sometimes I wish the band would give the instruments a bit more room and vary the tempo a bit more. Maybe I am not the target group for their art... maybe. Fans of occasionally faster played black metal, which comes with symphonic facets and thrash influences should give this release a try.
This releases comes with a professional printed booklet -- without lyrics -- and a normal CDr.


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When was the last laptop you dumped because the battery went dead? Frustrated because you need to check an email and your battery died on you while you were out? AC Ryan has a good solution for you… The MobiliT External Laptop battery is a 9 cell battery that comes with 6000mAh of power and a full set of power converters ensuring maximum compatibility with your mobile computers.
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a whole series of adaptors to ensure maximum compatibility with your laptop
Battery is charged via your laptop adaptor via the adaptors provided. With this battery, My IBM T43 can last me more than 8 hours on the go coupled with my stock 6 cell battery!


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Detroit Martin Luther King running back Dennis Norfleet was one of the top junior performers on the Midwest combine and camp circuit this off-season. The 5-9, 170-pound Norfleet carried over that s...
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Premier Medical Associates (PMA ) is seeking a BC/BE gastroenterologist to join the practice in Pittsburgh, Pennsylvania. This is a high volume practice with the opportunity to be busy immediately. In 2017, AMGA recognized Premier Medical Associates (PMA) with the Acclaim Award for quality care and the Optum Innovation Award. PMA is consistently recognized as a leader in value based care.
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Interested candidates please contact: Sarah Vincent Physician and Advanced Practice Recruiter (314)-236-4435 firstname.lastname@example.com www.cejkasearch.com


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Garden Design: Architecture and Decor
Look twice at the hardscaping and architectural features of show exhibits. When you see a gardenscape you like at a show, look more closely to see if there are any individual features you can adapt to fill a need in your garden.
While this al fresco kitchen is surely beyond my means, I can never have enough comfortable seating, or enough planting space. I really loved the stone benches backed by planter boxes, although creating one from stacked stones seems overly ambitious. However, I also liked the look of the stucco used on the other side of the exhibit. Putting those ideas together, I'm thinking that I could build a bench-planter from cinder blocks and then stucco its surfaces. If you've already got a bench you love, this exhibit might inspire you to finish it off with a cushion and pillows for a more elegant look.
This pergola design caught our eye because of its irregular shape. It wouldn't have occurred to me to build one that didn't have a rectangular footprint, but this would work brilliantly in a small, oddly shaped nook. Another demonstration garden used pavers to create a small, perfectly circular pathway, setting off an accent piece. A small card on the pathway told us this feature would be easy to recreate, as the pavers were available as a kit.
Look at the layout of pathways and patios in exhibits you find inviting. I'm drawn to informal exhibits with winding pathways with unknowns around every corner. Exhibits like that cram a lot of garden interest into relatively small spaces by avoiding straight pathways and direct sight lines. That's an idea you can take home to make the most of whatever garden space you have.
Garden Décor: Color Combinations and Decorating Schemes
Flower shows are great for getting ideas about colors and textures that work well together. The container plantings at the show were spectacular and often enormous, but in many cases you could imagine downscaled versions looking wonderful on your own patio. Just remember that you may have to do some substitution to get a similar effect. Specific plant combinations from the show are often not possible in an actual garden setting.
Other designs may not be really practical in a home version. However, when you see an idea you love, make a note of it. You may find a way to adapt some aspect of it to your home or garden. We admired a modern wall of flowing water studded with orchid blooms. I'm not sure how to achieve this formal design in a smaller, more maintainable way. But now I'm looking twice at vertical tabletop fountains.
A glowing golden room studded with sunflowers drew the interest of many flower show attendees. Vases of upright forsythia branches lined the walls to set the stage for a spectacular central arrangement of cherry blossoms illuminated by gold uplights. It wasn't a room I would want in my home, but I could imagine creating a similar effect for a reading corner, with sunflower-stenciled walls, a gold-shaded lamp over a simple upholstered chair, and a tall vase of ever-lasting pussy willow stems.
Garden Accents: Statues and Fountains
It's almost impossible to visit a garden show without deciding you must find a way to work more accents and decorative features into your own garden design. The Italian theme of the 2009 show translated to statues and fountains galore.
Many of the water features at the show were very simple, despite their enormous size. Even if you don't have room for a fountain the size of an Olympic lap pool, a simple rectangular trough with a trio of water spouts would be a great garden addition. On a smaller scale, salvias or liatris could be substituted for these big spikes of delphiniums, echoing the vertical fountains.
Successfully incorporating such a feature into a gardenscape involves more than simply finding a stable place to plop it down. Focal point pieces can seem like an afterthought unless they're integrated into your garden theme. A statue of St. Francis feeding the birds suits an informal, woodland setting, while a classical Greek bust would be more at home among formal trimmed hedges. Additional architecture such as a wall or an arch can work to elevate and frame a special piece.
Urns and planters provide wonderful accents to draw your eye. Not only do they set off the plants they contain, they also bring attention to the surrounding plantings. Notice how containers are placed in show exhibits. Large urns and taller planters work well to accent corners or define the curve of a garden path. Detailed, formal urns are most effective by themselves or placed in pairs to flank a pathway or other feature. Simpler containers can be grouped together, preferably in odd numbers.
A huge event like the Philadelphia Flower Show provides endless inspiration for garden design, garden décor, and garden accents. Find an exhibit you love, and focus on a feature that makes it work. Then downsize that idea, and bring it home!
If you go
The 2010 Philadelphia International Flower Show will run from February 28 through March 7 (Sunday to Sunday), with a "Passport to the World" theme. There's plenty of parking around the convention center, or you can take a bus there (look for special promotional tours). Weekdays are less crowded, of course, but the crowds do thin out after 3 or 4 in the afternoon even on weekends.
Don't miss the great food court area across the street! If you've never had a genuine Philly cheesesteak, it's a must.
Photos by Jill M Nicolaus. Move your mouse over images and links in the article for additional information.


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At Gemvara, we believe that the multistone september birthstone birthstone jewelry you wear should be precious because it's personal. Every beautiful Gemvara design is made just for you, in your choice of gems and precious metals. Your child's birthstone in sterling silver. Your favorite gem in yellow gold. Add your own meaning to every design, creating beautiful memories and brilliant reminders. It's jewelry for all life's occasions, from a birthday or engagement to your own personal milestones and celebrations. Gemvara uses only the highest quality materials to craft your multistone september birthstone birthstone jewelry with meticulous attention to detail. Our precious metals are recycled or responsibly mined. Our wide selection of multistone september birthstone birthstone jewelry by Jeet Palavwala, Carl Ansley, Ruzanna Davtian, and other talented designers means you can find jewelry that's just your style. Our gemstone jewelry designs are beautifully hand-crafted with each of the individual gems set by hand. The tradition of birthstone jewelry started with the Breastplate of Aaron: a priest's garment set with twelve gems. September's birthstone is sapphire, the ultimate blue gem. Gemvara also offers pink, yellow, and white sapphire for September birthstones to match every outfit. Clean your multistone september birthstone birthstone jewelry with mild dish soap: use a soft brush where dust collects. Gemvara's fine quality multistone september birthstone birthstone jewelry is designed to be passed down for generations to come, crafted by hand in the United States.
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Perfect attire for the master of the grill. Our fun Patio Daddio apron provides extra coverage for those inevitable barbecue sauce splashes. Two handy pockets in front are the perfect place to hold grill tools.


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Had to pass this onto you, one of those shell petrol station things but this one is actually worth having.
for 3.99 I had to have a dabble.
It's a camera tripod at the end of the day so worth the money for that alone. The 9 LED torch that screws on top is a definite bonus.
I was astonished to find it even came with batteries and all for less than the price of a pint of beer. This one will be a regular in the garage for me.


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SILVER SPRING, Md. (AP) — Montgomery County police say a northern Virginia woman has died as the result of a two-vehicle crash in Silver Spring.
Police say the vehicles collided early Sunday morning at the intersection of Tech Road and Route 29. Police say one car was facing a flashing red signal and tried to cross through the intersection, where it was hit by a vehicle that was driving through a flashing yellow signal.
The passenger who was killed has been identified as Marisol Angela Gutierrez, 29, of Woodbridge, Va. She was riding in the car that was facing the flashing yellow signal. She was pronounced dead at the scene.
The drivers of both cars were taken to hospitals and treated for non-life-threatening injuries.
(Copyright 2011 by The Associated Press. All Rights Reserved.)


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Welcome to the new Yvonne Strahovski website!
Welcome to your new website dedicated to the best things about Yvonne Strahovski.
I do the best to serve you with the latest pictures, news, interviews, videos about Yvonne Strahovski and all the people around her. You may know her as « Sarah Walker » in « Chuck« .
Now, she has finished shooting Chuck final season! You can also see her in many other movies : 'The canyon', 'Persons of Interest', 'The plex', 'Killer Elite' and soon : 'The mother's curse' renamed 'The Guilt Trip' and 'I Frankenstein'.!
She also joins the cast of the tv show Dexter on Showtime! You'll see her starting in October 2012 for several episodes!
Name:Yvonne Strahovski
Sent in her audition tape for the show « Chuck » (2007) in the United States while in the states doing an audition for two other shows (see special features of disk 3 of « Chuck » Season 1 « Chuck's World ». She sent in an audition tape while waiting to hear from the other two jobs. She was contacted by the producers of « Chuck » the next day to come in to « run lines » with Zachary Levi aka Chuck Bartowski/Charles Carmichael. The chemistry was undeniable and the producers of « Chuck » called her back 3 days later letting her know she got the job. She arrived to the United States and was cast as the leading lady on « Chuck » aka « Sarah Walker ».
• Ranked n°24 in Wizard magazine's Sexiest Women of TV list (March 2008).
Source : imdb.com
CLOUDS : Aaron Eckhart Adams Baldwin Australia Bill Nighy BioWare Bodypainting Chuck Chuck Bartowski Chuck series final Comic Con San Diego Deb Dexter exhibition Hannah McKay I Frankenstein Jennifer Carpenter Joshua E Gomez Killer Elite Mass Effect 3 Maxim Magazine Micheal C. Hall Miranda Lawson Miranda Otto Movie My mother's curese Ryan McParlin Sarah Lancaster Sarah Walker Sarha Walker Scott Krinsky sho SoBe Lifewater Stuart Beattie Top 100 Trailer TV Guide Vik Sahay Yvonne Strahovski Yvonne Strzechowski Zachary Levi
Yvonne-Strahovski.eu is an unofficial website. We are not affiliated with Yvonne Strahovski & any cast members, NBC, or Universal Studios, etc. No copyright infringement is intended. Please e-mail me at firstname.lastname@example.com if you like something taken down before taking any legal action.


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Welcome to Fodiator, range for e-drives The fuelcell-e-drive for sea and inland waters
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In October 2012 we introduced our new fuelcell e-drive to the media. In the presence of district chief executive Wägemann, Mr. Simon from the administrative district office gave an overview of the plans of Brombachsee special purpose association: a sea center, hydrogen filling station, hydrogen electrolysis. (Please see: Brombachsee project)
Now we are at the market in production with the first fuelcell-drive for everyone and every boat. We have systems from 1,1 to 10kW, which are 13.000-23.000€. It would be a pleasure, to make you an offer.
Boots-Charter Fahrland Ltd., a charter company offers a fishing and a leisure launch boat on Brombachsee starting June 2013, a luxurious house boat for holiday on water will follow. All boats will be fitted with Fodiator Green Fuelcell Drives. (Please see: Brombachsee project).
By the way … Fodiator is the genus name of a flying fish! (click here)


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Yet another handset that is showing its face via an FCC listing, this time it is the candybar styled P'9522 from Porsche Design. Sadly, the P'9522 is not offering as clean of a design as one would hope. Sure, it is a candybar handset, and one that (for the most part) seems to have a clean looking design. However in the case of the P'9522, it has a big fingerprint scanner front and center which takes away some of the good looks.
Otherwise it does have a nice big display, which adds some nice value. Aside from the external design, the US release of the P'9522 will only have GSM 850/1900 support, which means that unlike the European version, it will not have any 3G support. As expected with FCC listings, pricing information has not yet been revealed.
Read [FCC] Via [Engadget Mobile]


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Open Houses
There will be an open house bridal shower for Annie Dingman, bride to be of Matt Hoepner, on Saturday, April 27, at Faith Lutheran Church in Morris from 10 a.m. to noon. Annie is the daughter of Pat and Chuck Dingman of Morris. s17
In honor of Marcella Hansen's 90th birthday, Godly Women Out Of History will perform Sunday, May 5, at 2 p.m. at Trinity Lutheran Church in Cyrus. Lunch will follow. Let your presence be your gift. s18
There will be an open house wedding shower for Ashley Fehr, bride to be of Nate Knobloch on Thursday, May 2 from 6:30-8:30 p.m. at the Apostolic Christian Fellowship Hall, rural Morris. s17
There will be an open house baby shower for Teresa (Berg) Randall, on Saturday, April 27 from 2 to 4 p.m. in the Community Room of Grandview Apartments in Morris. All are welcome. s17
An open house wedding dance in honor of Leah Zierke and Chad Ver Steeg will be held Saturday, May 11, beginning at 8 p.m. at Lakeside in Glenwood. All are welcome. s18
There will be an open house bridal shower in honor of Mindy Janachovsky, bride to be of Scott Schmidt, Saturday, May 4 from 10:30 a.m. to 12:30 p.m. at St. John's Lutheran Church in Donnelly. s18
There will be an open house bridal shower for Ali Boomershine, bride to be of Neil Haugerud, on Saturday, May 4, from 9:30 to 11 a.m. at First Lutheran Church in Morris. s17
An open house bridal shower for Jill Reinert, bride to be of Brian Zimmerman, will be held Saturday, May 11 at Our Savior's Lutheran Church in Chokio, from 9:30 to 11:30 a.m. The couple is registered at Target, Macy's and Herberger's. s18
You are invited to a 90th birthday party for Shirley Henrichs on Sunday, April 28 at Trinity Lutheran Church, 735 2nd St. S.E., Milaca, Minn., with an open house from 1 to 5 p.m. for lunch. For those who are unable to attend, but would like to send birthday wishes: Elim Care & Rehab Center, 730 2nd St., S.E., Milaca, MN 56353. s17
There will be an open house bridal shower for Kendra Vipond, bride to be of Eric Weiss, on Saturday, May 11 from 9 to 11 a.m., upstairs at the Knotty Pine in Elbow Lake. Kendra is the daughter of Tim and Janelle Vipond of Wendell.