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Lack of Integrin ?1?1 Leads to Severe
Glomerulosclerosis after Glomerular Injury
Xiwu Chen,* Gilbert Moeckel,†Jason D. Morrow,‡
Dominic Cosgrove,§Raymond C. Harris,*¶
Agnes B. Fogo,†Roy Zent,*¶?and Ambra Pozzi*¶?
From the Department of Medicine,* Division of Nephrology, and
the Departments of Pathology,†Pharmacology,‡and Cancer
Biology,?Vanderbilt University Medical Center, Nashville,
Tennessee; the Veterans Affairs Hospital,¶Nashville, Tennessee;
and the Department of Cancer Genetics,§Boys Town National
Research Hospital, Omaha, Nebraska
Severity of fibrosis after injury is determined by the
nature of the injury and host genetic susceptibility.
Metabolism of collagen, the major component of fi-
brotic lesions, is, in part, regulated by integrins. Us-
ing a model of glomerular injury by adriamycin,
which induces reactive oxygen species (ROS) produc-
tion, we demonstrated that integrin ?1-null mice de-
velop more severe glomerulosclerosis than wild-type
mice. Moreover, primary ?1-null mesangial cells pro-
duce more ROS both at baseline and after adriamycin
treatment. Increased ROS synthesis leads to decreased
cell proliferation and increased glomerular collagen
IV accumulation that is reversed by antioxidants both
in vivo and in vitro. Thus, we have identified integrin
?1?1 as a modulator of glomerulosclerosis. In addi-
tion, we showed a novel pathway where integrin
?1?1 modulates ROS production, which in turn con-
trols collagen turnover and ultimately fibrosis. Be-
cause integrin ?1?1 is expressed in many cell types
this may represent a generalized mechanism of con-
trolling matrix accumulation, which has implications
for numerous diseases characterized by fibrosis.
(Am J Pathol 2004, 165:617–630)
Glomerulosclerosis, the process by which glomerular tis-
sue is replaced by extracellular matrix (ECM), is the final
common pathway for loss of functioning glomeruli. All
three major cell types that constitute the glomerulus con-
tribute to this process. Podocytes and endothelial cells
are likely critical for initiation of sclerosis; however, mes-
angial cells are the major contributor to progression.1The
normal glomerular response to injury is rapid synthesis of
ECM components (mainly collagens) that are remodeled
as the injured glomerulus undergoes repair. In glomeru-
losclerosis, ECM accumulation, particularly collagens, is
The synthesis of ECM is, in part, regulated by inte-
grins.2Integrin ?1?1, a major collagen receptor, is ex-
pressed in all cell types in the glomerulus.3,4This integrin
has been associated with renal disease and is overex-
pressed in proliferating mesangium in glomerulonephri-
tis.5,6In addition, anti-integrin ?1 antibodies reduced
scarring in rat models of glomerular injury by inhibiting
integrin ?1?1-dependent (VLA-1) leukocyte function with
consequent immune response dampening.7Despite
these results, one would expect that lack of integrin ?1?1
might predispose to increased glomerulosclerosis be-
cause integrin ?1?1 is critical for the support of cell
proliferation and survival on collagen substrata,8as well
as for sensing extracellular collagen levels and down-
regulating endogenous collagen synthesis.9
quently, loss of ?1?1 function results in increased colla-
gen expression9and an inability of ?1-null cells to
proliferate on collagenous substrata.8
To determine the role of integrin ?1?1 in mediating the
host response to renal injury, we compared the response
of wild-type and integrin ?1-null mice to adriamycin
(ADR)-induced nephropathy. This is a nonimmunologi-
cally mediated injury induced by generation of reactive
oxygen species (ROS) that is characterized by mild mes-
angial sclerosis in BALB/c mice.10–12We demonstrate
that ?1-null mice on the BALB/c background developed
severe glomerular injury, characterized by mesangiolysis
within 24 hours of ADR administration and consequent
relentless mesangial sclerosis. Excessive ROS produc-
tion was the major stimulus for the unwarranted excessive
synthesis of collagen by both ?1-null mice and primary
mesangial cells, at steady state levels and after treatment
with ADR. Excessive ROS-mediated collagen deposition
Supported by the National Institutes of Health (National Cancer Institute
grant R01 CA94849–01 to A.P.; grant DK59975 to G.M.; National Institute
of Diabetes and Digestive and Kidney Diseases Pediatric O'Brien Center
grants P50 DK44757 and DK56942 to A.B.F.; National Institute of Diabe-
tes and Digestive and Kidney Diseases O'Brien Center grant P50
DK39261-16 to R.Z., R.C.H., A.P., A.B.F.; grants GM15431, CA77839 and
DK48831 to J.D.M.); the Department of Veterans Affairs (an advanced
career development to R.Z. and a merit award to R.C.H. and R.Z.); the
American Heart Association (grant-in-aid to R.Z.); and the Burroughs
Wellcome Fund (clinical scientist award in translational research grant to
Accepted for publication April 29, 2004.
R.Z. is a Clinician Scientist of the National Kidney Foundation of the
Address reprint requests to Ambra Pozzi, Dept. of Medicine, Div.
of Nephrology and Hypertension, Vanderbilt University, Medical Center
North, B3109, 1161 21st Ave., Nashville, TN 37212. E-mail: ambra.pozzi@
American Journal of Pathology, Vol. 165, No. 2, August 2004
Copyright © American Society for Investigative Pathology
Page 2
could be ameliorated, both in vivo and in vitro, by treat-
ment with antioxidants. Together these results demon-
strate that integrin ?1?1 is a critical modulator of glomer-
ular injury and its functional loss leads to increased
sclerosis primarily by stimulating ROS production.
Materials and Methods
Mice and Experimental Procedure
All experiments were performed according to institutional
animal care guidelines. Wild-type and integrin ?1-null
BALB/c male mice (5 weeks old, ?20 g body weight)
received a single intravenous injection of ADR (10 mg/kg;
Sigma, St. Louis, MO) as described.10Mice were sacri-
ficed 0, 24, and 72 hours, or 1, 2, 4, 6, and 8 weeks after
ADR injection. Seven mice/genotype/treatment were
used for this set of experiments. Three independent ex-
periments were performed.
When antioxidants were used, mice were treated daily
with ?-tocopherol acetate (100 mg/kg daily i.p., Sigma)
and ascorbic acid (10 mg/ml in drinking water daily,
Sigma) beginning 7 days before ADR (ADR ? AOX
group) until sacrifice (1 week after ADR injection). Some
mice received ADR only (ADR group) and others only the
antioxidants (AOX group). One week after ADR injection
was chosen as an end point because matrix deposition
and hyalinosis were primarily observed in ?1-null, but not
wild-type mice, at this time (Figure 1). Ascorbic acid and
?-tocopherol acetate were used at the indicated doses
because they have been shown to ameliorate ROS-me-
diated injury in rodents.13,14Four mice/genotype/treat-
ment were used for this set of experiments. Two indepen-
dent experiments were performed.
Clinical Parameters and Morphological Analysis
Urine was collected 24 hours before sacrifice and urinary
albumin was measured by the ELISA Albuwell M test kit
(Exocel Inc., Philadelphia, PA) and expressed as mg/L.
F2-isoprostanes were measured in urine using stable iso-
tope dilution methods, gas chromatography negative ion
pressed as ng/mg urine creatinine. Blood was collected
immediately at sacrifice and serum creatinine was mea-
sured using a commercially available colorimetric kit
(Sigma) and expressed as mg/dL/g body weight.
Kidneys, removed immediately at sacrifice, were either
fixed in 4% formaldehyde and embedded in paraffin for
both morphological and immunohistochemistry analysis,
or depleted of medulla and frozen for mRNA analysis.
Paraffin tissue sections were stained with periodic acid-
Schiff or Jones' for the evaluation of glomerular injury.
Mesangial matrix expansion index (MMEI) was evaluated
in 100 glomeruli/kidney by a single blinded observer
(G.M.) and expressed by percentage as normal, mild,
and severe (0%, 1 to 30%, and ?30% of glomerular tuft
with visible expanded mesangial matrix, respectively).
Kidneys (n ? four or seven/genotype) were evaluated
and the MMEI expressed as mean ? SD for each group.
Electron Microscopy
Portions of renal cortex were fixed in 2.5% glutaraldehyde
in phosphate buffer. Samples were postfixed in OsO4,
dehydrated in ethanol, and embedded in resin. Thin sec-
tions were assessed for mesangiolysis, mesangial matrix
deposition, and podocyte injury. Foot process efface-
ment (FPE) was semiquantitatively assessed (percent of
effacement in 10 peripheral capillary loops/glomerulus
with a total of seven kidneys/genotype evaluated) and
expressed as mean ? SD for each study group.
Immunohistochemistry and
Immunohistochemistry on paraffin kidneys sections was
done using rabbit anti-mouse collagen IV antibodies (1:
100; Biodesign, Saco, ME), or rabbit anti-mouse prolifer-
ating cell nuclear antigen (PCNA) (1:100; Santa Cruz,
Santa Cruz, CA), followed by horseradish peroxidase-
conjugated goat secondary antibody to rabbit IgG (1:
200; Jackson Immunoresearch, West Grove, PA) and
Sigma Fast DAB chromogenic tablets (Sigma). The glo-
merular proliferation index was expressed as (number of
PCNA-positive glomerular cells/total number of glomeru-
lar cells) ? 100. Glomerular apoptosis was evaluated by
staining paraffin section with the Dead End colorimetric
terminal dUTP nick-end labeling (TUNEL) system (Pro-
mega, Madison, WI) using diaminobenzidine as the chro-
mogenic substrate. The glomerular apoptotic index was
expressed as (number of TUNEL-positive glomerular
cells/total number of glomerular cells) ? 100. Ten glo-
meruli/kidney were evaluated with a total of seven kid-
To determine collagen deposition by mesangical cells,
3 ? 104cells were plated for 48 hours on uncoated
four-well chamber slides in the presence of Dulbecco's
modified Eagle's medium (DMEM) containing 2% fetal
Figure 1. Integrin ?1-null mice develop severe glomerular injury after ADR injection. A: Creatinine (mg/dL/g) and albuminuria (mg/L) were measured from urine
or serum obtained from untreated or ADR-treated mice immediately at sacrifice. Data are presented as the mean and SD of seven wild-type (WT) and ?1-null (KO)
mice. Differences between untreated and treated ?1-null mice (*) or untreated and treated wild-type mice (**) or wild-type and ?1-null mice within the same
treatment group (?) were significant with P ? 0.05. B: Five-week-old male mice received a single intravenous injection of ADR (10 mg/kg) and kidneys were
evaluated at the time points indicated. Signs of early mesangiolysis (arrow), increased matrix deposition (arrowhead), and hyalinosis (*), were evident primarily
in the ?1-null mice 24 hours, 72 hours, and 1 week after ADR injection (periodic acid-Schiff). At later stages, increased ECM accumulation and sclerosis were more
severe in the ?1-null mice compared to their treated wild-type counterparts (Jones' staining). C: One hundred glomeruli/kidney from seven untreated and
ADR-treated mice were evaluated and the degree of matrix mesangial expansion (MMEI) scored and expressed as described in Materials and Methods. D:
Ultrastructural examination of kidney sections revealed early mesangiolysis (arrow), matrix accumulation (arrowhead), and FPE (*) primarily in the ?1-null mice
24 hours and 72 hours after ADR injection. FPE was also visible in wild-type mice 4 weeks after ADR injection, although it was much more severe in ?1-null. Scale
bar, 50 ?m. Original magnifications, ? 400 (B).
AJP August 2004, Vol. 165, No. 2
Chen et al
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Integrin ?1?1 in Glomerulosclerosis
AJP August 2004, Vol. 165, No. 2
Page 4
calf serum (FCS) and 0, 0.1, 0.5, and 1 ?mol/L ADR in the
presence of the antioxidants TEMPOL (10 ?mol/L) and
DPI (1 ?mol/L) (both from Sigma). The doses of TEMPOL,
a superoxide mimetic, and DPI, a specific NADPH oxi-
dase inhibitor, were chosen because they represent the
maximum dose able to inhibit ROS generation in wild-
type and ?1-null mesangial cells without cytotoxic effects
(data not shown). Immunofluorescence on acetone-fixed
cells was done using the rabbit anti-mouse collagen IV
antibodies described above (1:100) followed by fluores-
cein isothiocyanate-conjugated goat secondary antibody
to rabbit IgG (1:100; Jackson) and 2 ?g/ml 4,6-dia-
midino-2-phenylindole to visualize nuclei.
To determine collagen deposition by mesangial cells, 3 ?
105cells were plated on uncoated six-well plates in the
presence of DMEM containing 2% FCS and in the ab-
sence or presence of TEMPOL (10 ?mol/L) and DPI (1
?mol/L). After 48 hours, cells were scraped, suspended
in 50 mmol/L Hepes, pH 7.5, 150 mmol/L NaCl, 1% Triton
X-100, and centrifuged 10 minutes at 14,000 rpm. Cell
lysates were resolved by 5% sodium dodecyl sulfate-
polyacrylamide gel electrophoresis under reducing con-
ditions. Gels were either stained with Coomassie Blue to
evaluate equal loading, or transferred to Immobilon-P
membranes (Millipore, Billerica, MA). Membranes were
incubated with a rabbit anti-collagen IV antibody (Biode-
sign) and immunoreactive proteins visualized using a
peroxidase-conjugated goat anti-rabbit and an ECL kit
(Pierce, Rockford, IL).
RNA Analysis
Total RNA from renal cortices (n ? seven kidneys/geno-
type) was isolated using a single-step isolation kit (TRIzol;
Invitrogen, Carlsbad, CA). Ten ?g of total RNA was sep-
arated in formaldehyde-containing 1% agarose gels,
transferred to nylon membranes (Nytran Supercharge;
Schleicher & Schuell, Keene, NH), and hybridized with
32P-labeled cDNAs for mouse ?1(IV) collagen. Mouse
?2-tubulin was used for normalization. Collagen IV and
tubulin bands were quantified by densitometry analysis
using an Alpha Imager 2000 (Alpha Innotech, San Lean-
dro, CA) and collagen IV signal expressed as collagen
Isolation of Primary Mesangial Cells and
Proliferation Assays
Primary mesangial cells were isolated and characterized
as previously described.16For proliferation assays, 5 ?
103primary mesangial cells were plated in DMEM con-
taining 2% FCS on to 96-well plates coated with 10 ?g/ml
fibronectin or collagen IV (both from Sigma). In some
experiments, mesangial cells were cultured in DMEM
containing 2% FCS in the presence of different concen-
trations of ADR (0, 0.1, 0.5, 1, and 5 ?mol/L) alone or in
combination with TEMPOL (10 ?mol/L) and DPI (1 ?mol/
L). After 2 days the medium was replaced, the cells were
then pulsed for an additional 48 hours with3H-thymidine
(1 ?Ci/well) and processed as described.17Manual cell
counts paralleled the results of3H-thymidine incorpora-
tion (data not shown).
Flow Cytometry
A suspension of wild-type or ?1-null mesangial cells was
incubated with monoclonal antibodies of the appropriate
integrin (ie, anti-?1, -?2, -?5 and -?1 integrins all from
Pharmingen, San Diego, CA) (1:100 dilution), followed by
incubation with the appropriate secondary antibodies
(fluorescein isothiocyanate-coupled rabbit anti-rat or
hamster immunoglobulin) (1:100 dilution). Flow cytometry
was performed with a FACScan instrument (Becton Dick-
inson, Franklin Lakes, NJ). Cell suspensions incubated
with secondary antibody only were used as a negative
control for integrin expression. Two independent experi-
ments were performed in duplicate.
Adhesion Assay
Microtiter plates (96-well) were coated with fibronectin or
collagen IV at the indicated concentrations in phosphate-
buffered saline (PBS) for 1 hour at 37°C. Plates were then
washed with PBS and incubated with PBS containing 1%
bovine serum albumin for 60 minutes to block nonspecific
adhesion. One hundred ?l of single-cell suspensions
(5 ? 105mesangial cells/ml) in serum-free DMEM con-
taining 0.1% bovine serum albumin were added in qua-
druplicate to 96-well plates and incubated for 60 minutes
at 37°C. In some experiments, cell suspensions were
preincubated with anti-integrin antibodies (anti-?1, -?2,
and -?1 integrins) (10 ?g/ml final concentration) on ice for
30 minutes before the assay. Nonadherent cells were
removed by washing the wells with PBS. Cells were then
fixed with 4% formaldehyde, stained with 1% crystal vio-
let, solubilized in 20% acetic acid, and then read at 570
nm. Background cell adhesion to 1% bovine serum albu-
min-coated wells was subtracted from the values ob-
tained on ECM proteins. Two independent experiments
were performed in quadruplicate.
Detection of ROS Produced by Mesangial Cells
ROS in mesangial cells were measured using the sub-
strate dihydro-rhodamine (Molecular Probes, Eugene,
OR).18Briefly, 15 ? 104mesangial cells were plated in
six-well plates coated with 10 ?g/ml of fibronectin or
collagen IV in DMEM containing 10% FCS. After 24 hours,
cells were incubated for a total of 6 hours with DMEM
containing 2% FCS, 30 U/ml horseradish peroxidase, 2
?mol/L dihydro-rhodamine, and different concentrations
of ADR (0, 0.1, 1, and 5 ?mol/L) alone or together with
TEMPOL (10 ?mol/L) and/or DPI (1 ?mol/L). Cells were
then trypsinized, washed twice in PBS and the generation
of fluorescent rhodamine 123 was analyzed with a FAC-
Scan (?ex? 488 nm, ?em? 525 nm) as described.18
AJP August 2004, Vol. 165, No. 2
Chen et al
Page 5
Three independent experiments were performed in du-
Statistical Analysis
We used the t-test for comparisons between two groups,
and analysis of variance using Sigma-Stat software for
statistical differences between multiple groups. P ? 0.05
was considered statistically significant.
Integrin ?1-Null Mice Develop Severe
Glomerular Injury after ADR Injection
A single injection of ADR (10 mg/kg), previously shown to
induce renal injury in BALB/c mice,11,12was given to both
wild-type and integrin ?1-null mice on the BALB/c back-
ground. The wild-type mice developed an increase in
serum creatinine 2 weeks after ADR injection (Figure 1A),
which persisted until week 6 and started to recover by
week 8. These mice also developed significant protein-
uria that peaked 4 weeks after ADR but was still present
at week 8. In the ?1-null mice, the levels of serum creat-
inine and albuminuria were significantly worse when
compared to wild-type mice at all time points (Figure 1A).
Although integrin ?1-null mice develop normally and
have no overt renal phenotype,19their glomeruli are
smaller with reduced glomerular tuft area and evidence
of mild mesangial matrix accumulation (in 20% of glomer-
uli), when compared to wild-type controls (Figure 1, B
and C). Within 24 hours of ADR injection, kidneys from
?1-null mice, but not wild-type mice, showed evidence of
mesangial expansion, focal mesangiolysis with multiple
small electron-lucent mesangial lesions, and increased
FPE (31 ? 6.7% in the ?1-null versus 10 ? 6.5% in
wild-type; P ? 0.007; n ? 3) (Figure 1, B and D). By 72
hours the mesangial expansion and FPE had further in-
creased in ?1-null versus wild-type mice (67 ? 12% in the
?1-null versus 30 ? 10% in wild-type; P ? 0.01; n ? 3)
(Figure 1, B and D).
At 1 week glomerular matrix deposition and hyalinosis
became evident in ?1-null mice, but not in their wild-type
counterparts. Although wild-type kidneys showed mes-
angial expansion because of both mesangial cell prolif-
eration and increased mesangial matrix deposition (mea-
sured by MMEI) by week 4 as previously reported,11
mesangial expansion was far more severe in the ?1-null
mice at this time (Figure 1, B and C). By week 8 glomer-
ulosclerosis was well developed in the wild-type mice
and 40% of glomeruli demonstrated severe MMEI scores
(Figure 1, B and C). The pathology in the ?1-null mice
was significantly worse at the same time point and 60% of
glomeruli showed severe MMEI scores (Figure 1, B and
C). There was also worse interstitial damage of the kid-
neys in the ?1-null mice particularly evident at 4 and 8
weeks (Figure 1B).
Ultrastructural examination of kidneys from wild-type
mice showed segmental FPE 4 weeks after ADR admin-
istration, which became more widespread by week 8
(Figure 1D). In contrast, there was severe diffuse FPE
involving all of the capillary loops in the ?1-null mice at
week 4 and by week 8, the FPE was near complete
(Figure 1D). Although there was evidence of increased
mesangial ECM deposition in wild-type mice at 4 weeks
that progressed by week 8, the increase in ECM accu-
mulation in ?1-null mice was significantly greater at both
these time points.
ADR-Treated ?1-Null Mice Develop Mesangial
Expansion Primarily Because of Increased
Collagen IV Deposition
To determine the etiology of the mesangial expansion in
the ADR-treated mice we initially assessed the prolifera-
tion and apoptosis index in glomeruli of untreated and
ADR-treated mice. We found a significant increase in the
number of PCNA-positive cells primarily in the glomeruli
of wild-type mice. This increase was evident 1 week after
ADR injection (40 ? 7% in wild-type versus 25 ? 8% in
?1-null) and persisted throughout the duration of the
study period (Figure 2A). Because leukocyte infiltration
has been described in the ADR-induced injury mod-
el,11,12and integrin ?1-null mice show a defect in leuko-
cyte infiltration to the site of injury,20,21we assessed
whether the differences in number of PCNA-positive cells
could be because of lymphocyte and/or macrophage
infiltration. Both macrophage and lymphocyte infiltration
(assessed by staining kidney sections with anti-F4/80
and CD3 antibody, respectively) within glomeruli was
minimal in both genotypes and no significant differences
were observed at any time point examined (data not
shown). Because early mesangiolysis was observed
within 24 hours of ADR administration in the ?1-null back-
ground, we assessed apoptosis at this early time point in
both genotypes. As shown in Figure 2B, there was sig-
nificantly more apoptosis in the ?1-null mice compared to
their wild-type counterparts at early time points. At later
stages (weeks 1 to 8) apoptotic cells were difficult to
visualize in the glomeruli of both genotypes. Taken to-
gether, these results suggest that increased glomerular
proliferation of mesangial cells was not the etiology of the
excessive mesangial expansion in the ?1-null mice.
One of the characteristics of ADR-induced glomerular
injury is increased expression and accumulation of col-
lagen type IV in the glomerular tuft and Bowman's cap-
sule.10Because integrin ?1 is a primary receptor for
collagen IV19and is critical in down-regulating endoge-
nous collagen synthesis,9we analyzed and compared
collagen IV deposition in the glomeruli of ADR-treated
wild-type and ?1-null mice. Collagen IV mRNA and pro-
tein accumulation in ?1-null mice was increased 1 week
after ADR injection and persisted throughout the study
(Figure 2, C and D). Significantly less collagen IV synthe-
sis and deposition were observed in wild-type mice at all
time points examined (Figure 2, C and D). There was little
or no increase in glomerular collagen I deposition in either
the wild-type or ?1-null mice at any of the time points (data
not shown), suggesting that the primary cause of the in-
Integrin ?1?1 in Glomerulosclerosis
AJP August 2004, Vol. 165, No. 2
Page 6
Figure 2. Excessive collagen IV expression is the cause of the increased mesangial expansion in the ?1-null mice after ADR treatment. A and B: Kidney sections
from wild-type and integrin ?1-null mice were stained with anti-PCNA antibody (A) or with the Dead End colorimetric TUNEL system (B), and the percentage
of proliferating or apoptotic cells expressed as (number of PCNA-positive or apoptotic cells in the glomerulus/total number of cells in the glomerulus) ? 100. Ten
glomeruli/kidney were evaluated. Data are presented as mean and SD of seven kidneys/genotype. C: Collagen IV staining of kidney sections from wild-type and
integrin ?1-null mice after ADR treatment. Note the increased collagen deposition in the glomeruli of ?1-null mice at 4 weeks after ADR treatment. Maximum
difference between the two genotypes was observed at 8 weeks (anti-collagen IV). D: ?1(IV) collagen mRNA levels in renal cortices of wild-type and integrin
?1-null mice were normalized to the ?-2 tubulin mRNA levels and expressed as mRNA collagen IV/mRNA tubulin. Data are mean and SD of seven
kidneys/genotype. Increased collagen IV levels were observed in kidneys of both wild-type and ?1-null mice at 1 week after ADR treatment, but persisted only
in the ?1-null mice throughout the study period. *, **, and ? are as in Figure 1A. Original magnifications, ?400 (C).
AJP August 2004, Vol. 165, No. 2
Chen et al
Page 7
creased mesangial expansion in the ?1-null mice was be-
cause of excessive collagen IV expression.
Mesangial Cell Adhesion on Collagen IV Is
Integrin ?1?1-Dependent
Our in vivo observation that ADR leads to early mesangi-
olysis with increased apoptosis and consequent mesan-
gial expansion and glomerulosclerosis in ?1-null mice (Fig-
ure 1, B and D; Figure 2B) strongly suggested that
mesangial cells are a target for ADR.18We therefore iso-
lated primary mesangial cells to investigate the mecha-
nisms for the increased ADR-induced mesangial injury in
?1-null mice.
We initially determined the role of integrin ?1?1 on
mesangial cell adhesion to collagen IV. We established
that mesangial cells express both integrins ?1?1 and
?2?1 and integrin ?1-null cells do not compensate for
lack of ?1?1 by overexpressing the other major collagen
receptor ?2?1 (Figure 3A). Furthermore, the expression
of integrin ?5 (fibronectin binding receptor) and ?1 sub-
units was comparable in both genotypes (Figure 3A). We
then tested the ability of mesangial cells of both genotypes
to adhere to collagenous substrata. As demonstrated in
Figure 3B, no differences in adhesion were observed be-
tween wild-type and integrin ?1-null cells plated on fi-
bronectin (an integrin ?5?1-dependent ligand) (Figure 3B).
In contrast, despite expressing the integrin ?2 subunit, ?1-
null mesangial cells showed significantly reduced adhesion
on collagen IV compared to their wild-type counterparts
(Figure 3C), suggesting that integrin ?1?1 is the major
collagen IV-binding receptor in the mesangium. To further
verify that mesangial cell adhesion on collagen IV was me-
diated by integrin ?1?1, mesangial cells of both genotypes
were plated on collagen IV in the absence or presence of
anti-?1, -?2, and -?1 integrin antibodies. As shown in Figure
3D, adhesion of wild-type mesangial cells was significantly
impaired after incubation with anti-?1 or -?1 but not anti-?2
Taken together, these results suggest that mesangial cell
adhesion to collagen IV is an integrin ?1?1-dependent
ADR Reduces Mesangial Cell Proliferation that
Can Be Rescued by Antioxidants
We subsequently tested the ability of mesangial cells to
proliferate on both collagenous and noncollagenous sub-
strata, by assaying
type mesangial cells proliferated 1.7- and 2.2-fold more
than ?1-null cells on fibronectin and collagen IV, respec-
tively (Figure 4A). The results on collagen IV paralleled
our previous observation that collagens promoted growth
of wild-type cells, while they failed to promote that of
?1-null cells.8The differences in proliferation on fibronec-
tin (in the absence of the natural ligand of integrin ?1?1)
were surprising and suggested that at the time of the
assay (72 hours) either the ?1-null mesangial cells se-
creted enough collagen IV that could act as an inhibitory
3H-thymidine incorporation.17Wild-
ECM, or that lack of integrin ?1?1 decreased cell growth
by a mechanism that was autonomous of integrin ?1?1-
dependent cell-ECM interactions.
Because mesangial cells produce large amounts of
ROS18and ROS can modulate cell growth,22we deter-
mined whether alterations in ROS production might ex-
plain the differences in growth between wild-type and
?1-null mesangial cells on both fibronectin and collagen
IV. Using the fluorescent probe dihydro-rhodamine,18we
initially demonstrated that ?1-null mesangial cells synthe-
size more ROS than their wild-type counterparts, which
was independent of the substrate on which cells were
plated (Figure 4B). To assess whether increased levels of
ROS accounted for the decreased proliferation of ?1-null
mesangial cells, we determined the effect of the antioxi-
dants TEMPOL (a superoxide dismutase mimic) and di-
phenyleneiodonium (DPI, a NADPH oxidase-specific in-
hibitor)23,24on both ROS generation and mesangial cell
proliferation. Incubation with TEMPOL, DPI, or a combi-
nation of the two antioxidants significantly reduced ROS
Figure 3. Mesangial cell adhesion on collagen IV is integrin ?1?1-depen-
dent. A: Integrin expression by wild-type and integrin ?1-null mesangial
cells. Cells were incubated with antibodies to ?1, ?2, ?5, or ?1 integrin
subunits. Integrin expression is displayed by a shift in mean fluorescent
intensity compared with no primary antibody incubation using fluorescence-
activated cell sorting analysis. B and C: Primary mesangial cells (5 ? 104)
were plated onto 96-well plates coated with fibronectin (B) or collagen IV
(C) at the concentrations indicated for 1 hour in serum-free medium. Adher-
ent cells were then stained with crystal violet, lysed, and the OD measured.
Data represent the mean and SD of quadruplicate samples of 10 pooled
wild-type and integrin ?1-null mice. D: Mesangial cells were plated in the
presence or absence of blocking antibodies (10 ?g/ml) to the integrin
subunits indicated on collagen IV (5 ?g/ml) for 1 hour in serum-free me-
dium. Adhesion was then determined and expressed as described above.
Note that adhesion of mesangial cells to CIV is primarily integrin ?1?1-
mediated. Data are expressed as in B.
Integrin ?1?1 in Glomerulosclerosis
AJP August 2004, Vol. 165, No. 2
Page 8
generation in mesangial cells from both genotypes plated
on either fibronectin (data not shown) or collagen IV
(Figure 4B). The antioxidants rescued ?1-null mesangial
cell proliferation on fibronectin returning ?1-null cell
growth to wild-type levels (Figure 4A). However, the res-
cue of ?1-null mesangial cell proliferation on collagen IV
was only partial (Figure 4A). These data suggest that the
mechanism for decreased cell proliferation of integrin
?1-null mesangial cells on fibronectin is primarily related
to increased ROS production as the proliferative signal
from the ECM can be transduced by integrin ?5?1. How-
ever, when ?1-null mesangial cells are grown on collagen
IV, decreased proliferation is related to both increased
ROS production and loss of an integrin ?1?1-dependent
proliferative stimulus.8
To determine the effect of ADR on cell proliferation,
mesangial cells were cultured on fibronectin- or collagen
IV-coated dishes in the presence of increasing concen-
tration of ADR (Figure 5, A and B). As expected there was
a significant difference in proliferation between untreated
wild-type and ?1-null mesangial cells when plated on
both fibronectin and collagen IV. Increasing concentra-
tions of ADR reduced the percentage of cell proliferation
by the same amount (slope of the curves are approxi-
mately equal) in both phenotypes in the presence of low
concentrations of ADR (0.1 to 0.5 ?mol/L on fibronectin
and 0.1 to 1 ?mol/L on collagen IV). Treatment with
antioxidants rescued the proliferation of mesangial cells
of both genotypes to the same degree when they were
cultured on fibronectin (Figure 5A). In contrast, the res-
cue effects of the antioxidants on growth of the ?1-null
mesangial cells plated on collagen IV was only partial
(Figure 5B). To obtain direct evidence that decreased
proliferation in both wild-type and ?1-null mesangial cells
exposed to ADR was at least in part because of in-
creased ROS production, mesangial cells grown on fi-
bronectin (data not shown) or collagen IV (Figure 5C)
were incubated with different concentrations of ADR, and
ROS generation was measured. Although increased ROS
production was observed in both genotypes when ex-
posed to ADR, the ?1-null cells produced significantly
more ROS (Figure 5C) than their wild-type counterparts.
This increase in ADR-induced ROS generation was inhib-
ited after addition of TEMPOL and DPI either alone or in
combination (Figure 5D, only 5 ?mol/L shown). The res-
cue effect of antioxidants on proliferation was more evi-
dent when cells were treated with low (0.1 to 1 ?mol/L)
rather than high (5 ?mol/L) concentrations of ADR (Figure
5, A and B), despite almost complete inhibition of ROS
production by antioxidants in cells treated with 5 ?mol/L
ADR (Figure 5D). This result suggested that ADR might
prevent cell proliferation in a ROS-independent mecha-
nism when given at high concentrations for long periods
of time, as previously suggested.25Together these re-
sults show that ?1-null mesangial cells produce more
ROS in response to ADR compared to their wild-type
counterparts and the relative increase of ROS production
is proportional to the increased base line production of
the ?1-null mesangial cells. The direct correlation be-
tween ROS production and decreased cell proliferation
supports the notion that a significant increase in ROS
production by ?1-null mesangial cells plays a critical
contributory role in the control of mesangial cell prolifer-
ation. This mechanism is in addition to the lack of an
integrin ?1?1-dependent proliferative stimulus evident
when ?1-null mesangial cells are plated on collagen IV.8
ADR Induces Increased Collagen IV Synthesis
and Deposition in ?1-Null Mesangial Cells
Because ADR leads to increased ECM synthesis in vivo
(Figure 2, B and C),26we used immunofluorescence and
Western blot analysis to determine the effect of ADR on
collagen IV deposition/accumulation in wild-type and in-
tegrin ?1-null mesangial cells. At baseline ?1-null mes-
angial cells expressed significantly more collagen IV than
wild-type cells (Figure 6, A and B). Interestingly, although
treatment with antioxidants completely inhibited collagen
IV deposition in wild-type cells at baseline (Figure 6, A
and B) only a partial reduction in collagen accumulation
was observed in ?1-null mesangial cells exposed to an-
tioxidants (Figure 6, A and B). These results suggest that
although increased ROS generation by ?1-null mesangial
Figure 4. ?1-Null mesangial cells show reduced proliferation on both col-
lagenous and noncollagenous substrata. A: Primary mesangial cells (5 ? 103)
were plated on 10 ?g/ml of fibronectin or 10 ?g/ml of collagen IV in the
absence or presence of 10 ?mol/L TEMPOL (T), 1 ?mol/L DPI (D), or a
mixture of TEMPOL and DPI (TD). Incorporation of3H-thymidine was then
evaluated as described in Materials and Methods. Data represent the means
and SD of quadruplicate samples derived from 10 pooled wild-type and
integrin ?l-null mice. B: Mesangial cells were cultured on 10 ?g/ml of
fibronectin (FN)- or collagen IV (CIV)-coated dishes in the absence or
presence of 10 ?mol/L TEMPOL (T), 1 ?mol/L DPI (D), or a mixture of
TEMPOL and DPI (TD) together with 2 ?mol/L dihydro-rhodamine. Six
hours later, ROS generation was determined by FACS as described in Mate-
rials and Methods. Data represent the mean and SD of three experiments
performed in duplicates. *, **, and ? are as in Figure 1A.
AJP August 2004, Vol. 165, No. 2
Chen et al
Page 9
cells is a major contributor to increased collagen produc-
tion at baseline, there is also an integrin ?1-dependent/
ROS-independent mechanism that controls collagen depo-
sition/accumulation. After ADR exposure, both genotypes
showed increased accumulation of collagen IV (Figure 6A),
although ?1-null mesangial cells produced considerably
more collagen IV than wild-type cells. Addition of antioxi-
dants to both wild-type and ?1-null mesangial cells mark-
edly reduced collagen deposition after ADR treatment (Fig-
ure 6A) suggesting that ADR induces ECM deposition in a
ROS-dependent manner.9
In Vivo Treatment with Antioxidants Ameliorates
ADR-Induced Glomerular Injury
Because antioxidants rescued ?1-null mesangial cell
proliferation and collagen deposition in vitro (Figures 5
and 6), we investigated their effects in vivo. Pretreatment
of ?1-null mice with vitamins C and E significantly de-
creased ADR-induced matrix deposition, collagen IV ac-
cumulation, and MMEI (Figure 7; A to C). Despite this
improvement, increased collagen deposition and mesan-
gial expansion was still evident in ?1-null compared to
wild mice (Figure 7, A to C; ADR ? AOX group), suggest-
ing that lack of integrin ?1?1 per se plays a role in the
host response to injury. To demonstrate a correlation
between increased ROS production and the degree of
glomerular injury we measured F2-isoprostanes as a bi-
omarker of free radical oxidative stress.27Urinary (Figure
7, D and E) and isolated kidney (data not shown) F2-
isoprostanes were significantly increased both at base-
line and after ADR administration in the ?1-null mice
when compared to their wild-type counterparts. In vivo
treatment with antioxidants (which ameliorated glomeru-
Figure 5. ADR reduces mesangial cell proliferation that can be rescued by antioxidants. A and B: Mesangial cells were plated on 10 ?g/ml fibronectin (A)- or
10 ?g/ml collagen IV (B)-coated dishes in the presence of ADR at the concentration indicated in the absence of presence of mixture of 10 ?mol/L TEMPOL and
1 ?mol/L DPI (TD).3H-thymidine incorporation was then evaluated as described in Materials and Methods. Data represent means and SD of quadruplicate
samples of mesangial cells derived from 10 pooled wild-type and integrin ?l-null mice. C and D: Wild-type and ?1-null mesangial cells were plated on 10 ?g/ml
collagen IV in the presence of ADR at concentrations indicated (C) or with 5 ?mol/L ADR and 10 ?mol/L TEMPOL (T), 1 ?mol/L DPI (D), or a mixture of TEMPOL
and DPI (TD) (D). ROS generation was determined after incubation with 2 ?mol/L dihydro-rhodamine for 6 hours as described in Materials and Methods. Data
represent the mean and SD of three experiments performed in duplicates. *, **, and ? are as in Figure 1A. Differences between ADR-treated ?1-null and ADR ?
antioxidant-treated ?1-null cells (#) or wild-type and ?1-null cells within the same ADR ? antioxidants group (##) were significant with P ? 0.05.
Integrin ?1?1 in Glomerulosclerosis
AJP August 2004, Vol. 165, No. 2
Page 10
lar injury) decreased F2-isoprostanes production in both
ADR-treated wild-type and ?1-null mice. These results
strongly suggest that, in addition to the lack of integrin
?1?1 per se, increased ROS production by ?1-null mice
plays a significant contributory role in glomerular abnor-
malities at baseline, as well as in the severe acute injury
and consequent fibrosis after ADR administration.
The role of integrin ?1?1 in the host response to renal
injury is unclear. Functional inhibition of integrin ?1?1
(VLA1) on leukocytes decreases glomerular damage in
models of crescentic glomerulonephritis7and Alport's
syndrome.28In contrast, integrin ?1?1 is critical for
down-regulating collagen synthesis,9
functional loss could predispose the host to increased
sclerosis after injury. In our study we demonstrated that
injury after ADR injection was much worse in integrin
?1-null mice compared to controls. This increased injury
was primarily ascribed to excessive ROS production by
?1-null mesangial cells both at baseline as well as in
response to ADR. Treatment with antioxidants signifi-
cantly decreased collagen IV synthesis in ?1-null mes-
suggesting its
Figure 6. Collagen deposition in wild-type and integrin ?1-null mesangial cells. A: Mesangial cells were plated on uncoated chamber slides and treated for 48
hours with ADR at ?mol/L concentrations indicated in the absence (left) or presence of 10 ?mol/L TEMPOL and 1 ?mol/L DPI (right). Indirect immunofluo-
rescence was then performed to evaluate collagen IV synthesis and deposition. ?1-Null mesangial cells synthesize more collagen IV than their wild-type
counterparts and this synthesis is enhanced after ADR treatment. Although treatment with antioxidants (AOX) (right) reduced collagen deposition in both
untreated and ADR-treated cells, ?1-null mesangial cells still produced more collagen IV than their wild-type counterparts. B: Cell lysates were prepared from
untreated or antioxidant-treated mesangial cells cultured as described in A and Western blot analysis was performed using anti-mouse collagen IV-specific
antibodies. Note that ?1-null mesangial cells produce more collagen IV than their wild-type counterparts, and treatment with antioxidants only partially rescues
collagen synthesis. In contrast, antioxidant treatment completely inhibited collagen synthesis in wild-type cells, paralleling the results obtained with immuno-
fluorescence (A). Note that collagen IV is detectable in wild-type cells only when 100 ?g of total cell lysate were used for analysis. Scale bar, 20 ?m (A).
AJP August 2004, Vol. 165, No. 2
Chen et al
Page 11
angial cells and ameliorated renal injury in ?1-null mice in
vivo. These results confirm that the major changes seen
both in vivo and in vitro are dependent on increased ROS
generation and are not simply mediated by impairing the
ability of the host to down-regulate collagen. Thus, lack of
integrin ?1?1 may be a critical susceptibility determinant
in glomerulosclerosis by making the glomerulus far more
vulnerable to acute injury via the regulation of ROS pro-
duction by mesangial cells.
Integrin ?1-null mice showed a much worse response
to the injury at all time points assessed and many of the
?1-null mice kept longer than 8 weeks after ADR injection
were severely nephrotic and died from renal failure (data
not shown). The significant increase in glomerulosclero-
sis, decreased glomerular cell proliferation, and in-
creased apoptosis in the ?1-null mice are consistent with
the findings that integrin ?1?1 is a negative regulator of
collagen synthesis at both transcriptional and transla-
tional levels9and is critical to support proliferation on
collagenous substrata.8Our data are however in contrast
to the observation that blocking integrin ?1?1 leads to
reduced glomerular scarring during crescentic glomeru-
lonephritis.7The crescentic glomerulonephritis model is
an immunologically mediated injury induced by exposure
to anti-glomerular antibodies. Integrin ?1?1 is highly ex-
pressed on leukocytes, in addition to renal cells, and it is
likely that anti-?1 antibodies prevent leukocyte infiltration,
thus ameliorating glomerular scarring during crescentic
glomerulonephritis. Consistent with this data, deletion of
integrin ?1 results in decreased accumulation of macro-
phages in the kidneys of Alport mice and delays disease
progression.28,29Decreased leukocyte infiltration, with
consequent dampening of the inflammatory response
and tissue damage, has been also observed in integrin
Figure 7. In vivo treatment with antioxidants ameliorates ADR-induced renal injury. Five-week-old male mice (n ? four/genotype) received ADR, antioxidants
(AOXs), or a combination of ADR and AOX as described in Materials and Methods, and kidneys were analyzed 1 week after ADR injection. The increased ECM
deposition, determined by both Jones' (A) and collagen IV (B) staining, evident in ADR-treated ?1-null mice, was significantly ameliorated by administration of
AOXs. C: MMEI in glomeruli from ADR-, AOX-, and ADR ? AOX-treated mice was evaluated as described in Materials and Methods. D and E: F2-isoprostanes
were measured in the urine as described in Materials and Methods and expressed as ng/mg urine creatinine. Values represent the mean and SD calculated for
four samples/treatment. F2-isoprostanes levels in the urine of untreated ?1-null mice were significantly increased compared to their untreated wild-type
counterparts (P ? 0.05). Pretreatment with AOXs significantly reduced the excretion of this biomarker for oxidative stress in the ?1-null mice (P ? 0.05). ADR
injection led to increased excretion of F2-isoprostanes in both wild-type and integrin ?1-null mice (*, P ? 0.05 relative to untreated mice), although it was
significantly higher in the latter group, starting 1 day after ADR injection (**, P ? 0.05 between wild-type and ?1-null mice within the same group). AOX treatment
in ADR-treated mice significantly reduced F2-isoprostanes excretion in both genotypes (?, P ? 0.05 between ADR-treated and ADR ? AOX-treated mice). Original
magnifications, ?400 (A, B).
Integrin ?1?1 in Glomerulosclerosis
AJP August 2004, Vol. 165, No. 2
Page 12
?1-null mice in other model systems such as contact
hypersensitivity and arthritis.21In the ADR mouse model
we did not observe any discrepancy in leukocyte infiltra-
tion between the ?1-null mice and wild-type controls
suggesting that the differences in response to injury was
mediated by cells found within the glomerulus.
Significantly increased renal accumulation of collagen
IV in the glomerulus (Figure 2C) and collagen I in the
interstitium (data not shown) in integrin ?1-null mice is
consistent with the anatomical distribution of these ECM
proteins. Integrin ?1?1 negatively regulates collagen
synthesis and increased collagen I accumulation is ob-
served in skin19as well as bone calluses of integrin
?1-null mice.30Accumulation of collagen in bones30and
kidneys (present study) of ?1-null mice leads to scarring,
which is unlike that observed in the skin of these animals
after wounding.19In the wounded skin, dermal collagen
production is compensated for by increased synthesis of
MMPs, namely MMP13,19MMP2, and MMP9.17In con-
trast, no compensatory increase in MMP expression is
observed in the kidneys (data not shown) or bones.30
Thus, the severity of glomerulosclerosis observed in
ADR-treated integrin ?1-null mice is explained by in-
creased collagen synthesis with no compensatory up-
regulation of MMPs. These results suggest that the re-
sponse to injury in integrin ?1-null mice is organ- and
possibly even cell type-specific.
The rapidity of the renal injury in ?1-null mice, charac-
terized by mesangiolysis within 24 hours and accumula-
tion of mesangial ECM by 72 hours after ADR injection,
was remarkable. Mesangiolysis is a sign of severe injury
that is associated with loss of glomerular structure estab-
lished and maintained by interactions of mesangial cells
with the adjacent ECM. In ?1-null mice at baseline there
are probably sufficient compensatory mechanisms for
the glomeruli to function normally despite impairment of
?1?1 integrin-dependent interactions with surrounding
ECM. However, after injury, this compensation may be
insufficient to maintain tissue integrity, resulting in loss of
attachment of mesangial cells to the mesangial matrix
resulting in mesangiolysis and subsequent accelerated
ECM deposition during the repair process. A similar, but
much less marked phenotype occurs when integrin ?8-
null mice are exposed to mechanical stress induced by
hypertension.31Like integrin ?1?1, integrin ?8?1 ap-
pears to be critical for normal formation of the mesangium
where it interacts with its ECM ligands.31
We demonstrated that decreased growth of ?1-null
mesangial cells on collagen IV was because of both an
increase in ROS production as well as lack of integrin
?1-dependent mesangial cell-ECM interactions. We pre-
viously demonstrated that integrin ?1?1 is required to
support cell proliferation on collagens via activation of the
Shc/Grb2/MAPK pathway and failure to activate this path-
way in integrin ?1-null cells resulted in decreased prolif-
eration of fibroblasts plated on collagenous substrata.8
Presumably a similar mechanism is occurring in the inte-
grin ?1-null mesangial cells grown on collagen substrata,
which would explain why addition of antioxidants resulted
in only a partial rescue of ?1-null mesangial cell growth
on collagen. In contrast antioxidants completely rescued
proliferation of ?1-null mesangial cells on fibronectin. In
this scenario other integrins (such as ?5?1) interact with
ECM and probably result in activation of the Shc/GRB2/
MAPK pathways.8,32A schematic illustration of how cell
growth might be regulated in ?1-null mesangial cells is
proposed in Figure 8A. The mechanism by which integrin
?1?1 controls ROS generation is currently unknown, how-
ever, integrin ?2?1, another major collagen binding re-
ceptor, is a positive modulator of NADPH-regulated ROS-
dependent cellular functions. When integrins ?1?1 and
?2?1 are expressed on the same cell, ?1?1 negatively
regulates many integrin ?2?1-dependent functions, such
as collagen and MMP synthesis.9,33Therefore, lack of
integrin ?1?1 may result in uncontrolled ?2?1-mediated
Figure 8. Regulation of cell proliferation and collagen synthesis in ?1-null
mesangial cells. Schematic illustration of how cell proliferation (A) or colla-
gen synthesis (B) might be regulated in ?1-null mesangial cells. Integrin
?1?1 is the only collagen-binding receptor able to support cell proliferation
and down-regulate endogenous collagen synthesis on collagens via activa-
tion of the Shc/Grb2/MAPK pathway.8,33In the absence of integrin ?1?1,
cells plated on collagen substrata develop up-regulated synthesis of ROS and
decreased activation of the Shc/Grb2/MAPK pathway that could lead to
reduced cell proliferation (A) and increased collagen synthesis (B). When
cells are plated on collagen, addition of antioxidants (AOXs) could only
partially rescue ?1-null mesangial cell growth (A) or partially decrease
collagen synthesis (B) because of the persistent lack of MAPK activation. In
contrast, treatment with antioxidants could completely rescue proliferation of
?1-null mesangial cells on fibronectin (A) as on this matrix, other integrins
(ie, ?5?1) could promote cell proliferation via activation of the Shc/Grb2/
MAPK pathways.8,32
AJP August 2004, Vol. 165, No. 2
Chen et al
Page 13
ROS production at baseline, which is further accentuated
by exposure to ADR both in vivo and in vitro.
ROS mediate numerous physiological cellular functions
at low levels but they become cytotoxic at higher concen-
trations. Increased ROS production by ?1-null mice and
mesangial cells, at baseline and in response to ADR, results
not only in decreased cell growth, but also up-regulated
collagen synthesis. The observation that treatment with an-
tioxidants only partially down-regulate collagen synthesis in
the ?1-null cells, suggests that although increased ROS
generation by ?1-null mesangial cells is a major contributor
to increased collagen production, there is also an integrin
?1-dependent/ROS-independent mechanism that controls
collagen synthesis. This would agree with our previous find-
ings that integrin ?1 modulates collagen synthesis at both
transcriptional and translational levels9(Figure 8B). ROS
generation is directly correlated to many diseases charac-
terized by fibrosis in which they induce up-regulation of
profibrotic factors, such as transforming growth factor-?34
and connective tissue growth factor.35Therefore, increased
ROS production may induce synthesis of these growth fac-
tors that contribute significantly to the severe scarring phe-
notype seen in the ?1-null mice after ADR-induced injury.
In conclusion, we have demonstrated that lack of inte-
grin ?1 predisposes mice to severe acute damage and
excessive sclerosis after nonimmunologically induced re-
nal injury. This response to the insult is mediated by both
the loss of direct interactions of integrin ?1?1 with its
ligand collagen IV, as well as increased production of
ROS. Integrin ?1?1 may be regarded as a potent modifier
for injury-induced glomerulosclerosis in particular, and
possibly organ fibrosis in general.
We thank Ellen Donnert and Catherine Allen (Veterans
Administration Flow Cytometry Unit) for excellent techni-
cal assistance and Dr. Billy Hudson for his critical review
of the manuscript.
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AJP August 2004, Vol. 165, No. 2
Chen et al


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-Family Guy, Toy Story
It's times like this I wish I was a Transformer. -Toy Story
LOL! Seriously though, just stop. -Disney
Princess Merida from "Brave" in "The Expendables 2" Nemo from "Finding Nemo" in "Piranha 3DD" Buzz and Woody from "Toy Story" in "Magic Mike" Wall-E from "Wall-E" in "The Amazing Spider-Man" Carl from "Up" in "The Dark Knight Rises"

subset: commoncrawl


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nice but small
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Wedding Invitation
Signature of Love Rectangle Invitations The invitations were beautiful and arrived quickly. I wish there was an option to customize the back though.


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This Grade II listed building was Harrogate's first hotel and enjoys a peaceful location in landscaped grounds, close to the town centre. It provides spacious, well-equipped accommodation. Public area...More about BEST WESTERN PLUS Cedar Court Hotel
Sitting just a few miles from St Andrews, overlooking the rugged Fife coastline and the championship golf courses, The Fairmont is situated on a 520-acre estate. There are spacious bedrooms and bathro...More about Thorpe Park Hotel & Spa
Modern, budget hotel offering comfortable accommodation in bright and practical bedrooms. Breakfast is self-service and dinner is available in the restaurant. ...More about Ibis Budget Leeds Centre
Situated south of the city, this hotel offers attractively styled, modern, comfortable bedrooms. The restaurant offers an extensive range of food using fresh, local produce; lighter snack meals are se...More about BEST WESTERN Bradford Guide Post Hotel
Modern, budget hotel offering comfortable accommodation in bright and practical bedrooms. Breakfast is self-service and dinner is available in the restaurant. ...More about Ibis Budget Bradford
The Woodman Inn is extremely popular with locals. The popular bar offers a wide selection of real ales, lagers and draught ciders. Bedrooms have been recently refurbished and are comfortable and compr...More about Woodman Inn
An attractive Georgian hotel situated in its own grounds. All the bedrooms are individually designed to a high specification; some are in the old house and some in the converted stables set around a g...More about BEST WESTERN York Pavilion Hotel
This country pub and lodge is convenient for Harrogate, with its tourist attractions, shops and tearooms as well as the Conference Centre. It is also well located for visiting picturesque Knaresboroug...More about Innkeeper's Lodge Harrogate (East)
Conveniently situated only a few minutes' walk from the main terminal at Robin Hood Airport, this purpose-built hotel is a good base for both the business and the leisure traveller. The air-conditione...More about Ramada Encore Doncaster Airport
This Innkeeper's Lodge is a 19th-century Victorian property idyllically located in the Yorkshire Dales, with stunning views over Wharfedale. Perfect for walkers, browsing the spa town of Ilkley, close...More about Innkeeper's Lodge Ilkley


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|Shop By Age
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|H38.1 x W52.8 x D13cm
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Returns for UKIf for any reason you are not satisfied with your George Clothing or George Home product, simply return it within 100 days with proof of purchase for an exchange or refund. George branded products only. Excludes watches, jewellery, hair accessories, part assembled flat pack furniture and mattresses. The 100 Day Guarantee only applies to George branded products. Please refer to our terms and conditions for details of our returns and refund process for non-George branded products. Your statutory rights are not affected. UK Mainland stores only. Excludes franchises.
Rated 4.8 out of 5 by 72 reviewers.
Rated 5 out of 5 by amrobe A Great Walker I bought this as a present as my baby has one of these and I think it's great. The front comes away from the frame so pre-walkers can sit on the floor with it and press the buttons to hear the songs and make lots of noise with the little discs and flaps. It's durable too, even though my daughter is now 18 months old and walks just fine she still loves to push this around the house! Plus she can still learn from it through the songs it plays and words it says, she also likes playing with the phone. All in all I don't have anything negative to say about this product, I'm very happy I chose this walker as it's lasted well and still looks as good as new. Even though my daughter can walk she still gets alot of use out of this toy. 09 December 2011
Rated 5 out of 5 by Blue71 An excellent toy I had this given to me when my little boy was about 6 months old and he played with it loads. It's got lots to amuse little ones with and the activity panel can easily be removed from the walker so that your child can sit and play with it. I used to pop it in my changing bag to take out with us as one of his favorite toys. When he came to take his first steps, this walker was great. It was strong and sturdy and really helped him to get around. I therefore took the opportunity to buy one of these walkers for my cousin who recently had a baby as I know she will get lots of use from it! It's an excellent product. 06 January 2013
Rated 5 out of 5 by nuthill a very good buy. The VTech walker is very sturdy and helps my baby keep his balance while attemting to walk. The toys on the walker keep my baby entertained and interested, the plastic makes it easy to keep clean, as an aid to walking it is very practicle. 11 May 2010
Rated 5 out of 5 by Vicky21 Great purchase I purchased this item for my granddaughter who had just turned 1 years old. She loved!! the bright colours engaged her interest and the sound quality if wonderful. She loves pushing it about and answering the phone and playing with all the activities. I would highly recommend this item for any young baby just starting to walk. I can see hours of funny ahead with this purchase. 19 December 2011
Rated 5 out of 5 by yummumto2 One of my best buys ever! This baby walker is brilliant.My daughter isn't even at the stage where she can use it to walk but she always want to sit and play with the activity part of it that you can take off.So I can't see why she will not use it when she is able to start walking with it.Brilliant Item great value for money. 06 July 2011
Rated 5 out of 5 by dorsetvillager Excellent value for money Detachable so baby can play with it on the floor and walk along. Lots of different things for baby to play with - she really enjoys it! 19 December 2012
Rated 4 out of 5 by eddieteddy Great toy for learning and playing We bought this walker as it seemed to combine playing with helping our son to get on his feet and develop his walking. Firstly the entertainment value of the activity centre is superb. He loves it. It's a bit random (incorporating a farmyard, a piano and a phone) but it has a lot on offer and he always has something to do. The walkability is ok. It can be a bit scooty i.e.when our son pushes forward it can just go shooting off leading to him falling flat on his face (it's especially scooty on hard floors). But this is an issue on most walkers so we just need to be there to support him to avoid this sort of problem. I'm sure once he gets a bit more stable on his feet the walker will perform better. The quality is ok. Its all plastic but feels quite sturdy so hopefully it will last a while. All in, we're happy with it and for the entertainment value of the activity centre alone this is well worth the money. 02 January 2013
Rated 5 out of 5 by GREATGRAN63 GREAT TOY I purchased this walker for my Great Grandson who is 6 months old. Although he is a little young for it at the moment, the bright colours and the activity centre were immediately a hit with him. This should last a while, as when a little older he will be able to use the baby walker as well as the activity centre. All in all a worthwhile purchase and excellent value for money 27 December 2012
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The Huddle
High School sports in Southeast Texas
Posts filtered on Category
LIVE COVERAGE: West Brook Vs. Tyler Lee High School
Join Beaumont Enterprise sports writer, Jared Ainsworth, as he provides live coverage of the West Brook Vs. Tyler Lee High School football game Saturday evening from the Carroll A. "Butch" Thomas Educational Support Center in Beaumont. West Brook Vs. Tyler Lee high School [Read More]
Eddins named Built Ford Tough Player of the Week
Kelly quarterback Dakota Eddins was one of six players statewide named to the Built Ford Tough Texas High School Players of the Week The award came after Eddins passed for 428 yards and four touchdowns in Kelly's 42-31 win at Liberty last Friday. For the season, Eddins has passed for nine touchdowns and 1,129 yards at […] [Read More]
Reyes will be a game time decision
West Brook head coach Craig Stump says it's too early in the week to know whether or not quarterback Bruce Reyes will start Saturday's game at home against Tyler Lee. Reyes left the game with a shoulder injury with around 9 minutes and 36 seconds to play in the second quarter in Saturday's 45-21 loss […] [Read More]
Texas Coach Mack Brown issues statement on Garrett
Texas head coach Mack Brown talked about Reggie Garrett in his press conference today. Garrett had a mural of a University of Texas football helmet on the wall in front of his bed. "We send our thoughts and prayers to the family of Reggie Garrett. He and his family were very close friends with Earl […] [Read More]
Earl Thomas reacts to Garrett's death
The Denver Post had a story today about Earl Thomas' reaction to West Orange-Stark quarterback Reggie Garrett's tragic death on Friday night. Thomas wore No. 12 at West Orange-Stark and that number was retired. Thomas gave his permission for Garrett to wear his retired number. Here is the story about Thomas' reaction: http://www.denverpost.com/broncos/ci_16119646. [Read More]
WO-S will not practice this week, Kirbyville game still on
West Orange-Stark head coach Dan Hooks said the team will not practice this week following quarterback Reggie Garrett's tragic death on Friday night. The scheduled game with Giddings this Saturday has been canceled. The team returns to practice a week from Monday and will play Kirbyville on Saturday, Oct. 2 "We're going to use this […] [Read More]
West Orange-Stark's next game canceled
Interim West Orange-Cove Consolidated ISD Superintendent Bill Conway said West Orange-Stark's game Saturday, Sept. 25 has been canceled. The Mustangs were scheduled to play Giddings in Conroe. The district has not made a decision on the Kirbyville game in two weeks. [Read More]
LIVE COVERAGE: West Brook Vs. Katy High School
Join Beaumont Enterprise sports writer David Henry as he provides live web coverage of Saturday night's high school football game between No. 2 Katy High School and No. 6 West Brook High School from the Carrol A. "Butch" Thomas Educational Support Center. Kick off is at 6:00 p.m. West Brook Vs. Katy High School [Read More]
Katy football player returns to field after bout with cancer
The Houston Chronicle's Sam Khan wrote a feel-good story about Katy defensive end Nick Narcisse. Narcisse, who was thought to be a top college prospect after his junior season, was diagnosed with cancer in February and lost 40 pounds while battling it. He was declared cancer free in August and will take the field for […] [Read More]
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The food was better than average. The staff friendly, but the service was slow. Very slow. Fortunately, we had a lot of time. The environment modest. For smokers, they have "sheesha." And they have a bar.
If you own or manage Sabrina's Ethiopian Restaurant, register now for free tools to enhance your listing, attract new reviews, and respond to reviewers.


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Wedding decorations, party supplies, home décor & craft supplies at 20-70% off. Save On Crafts brings you classic and trending fashions.
Bring glamorous, sparkling detail to your Christmas tree or fanciful tablescape with this French cut glass jewel. The faceted Hanging Crystal will send dazzling light in every direction, and compliment lavish surroundings. For a stunning arrangement, string this gem on a fish line loop, and hang in front of a stringed light bulb on your Christmas tree. This piece is 3" tall and 2" tall, and is equipped with a small hole to hang.
Make a dramatic display using natural branches.


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- Lawmaker says license plate readers a privacy threat
- Bryant not the first to feel impact of free agency rules
- State Roundup: Parents' group calls for standardized test opt-out bill
- Hononegah Mack: 'The best woman in the county'
- The tip of the iceberg: Human trafficking in America
- State Roundup: House passes proposal to fill current fiscal year budget gap
- 'Hogs streak hits 4 as race tightens
- Neighborhood feel key for Rural on Tap
- TRRT March 25-31 | Online Edition
- State Roundup: Plaintiffs join Rauner on fair share case
Upper Palette: Tom Macs new album
Upper Palette: Tom Macs new album
By Molly Fleming, Staff Writer
Local musician Tom Mac will be releasing a solo CD this year titled Upper Palette. Originally set for release Dec. 31, 2002, the album was held a few weeks for further polishing and will be available for listeners sometime in January. The jazz influenced folk nine-song disk includes original compositions by Mac as well as jazz standards and has been in the works since 2001.
Mac is well known in Rockford for his folk/pop groups that frequently perform at Café Patou, Paragon and other local venues that host jazz and jazz-influenced music. Mac also has the ability to bring in well-known musicians of great status to perform with him, such as saxophonist Marc Colby and drummer Paul Wertico, who have both performed with him in the summer of 2002 in Rockford.
The recording of the new CD took place in Chicago, Austin, Texas, and Sedona, Ariz.. Artists included on Upper Palette are world-renowned guitarist Stanley Jordan, seven-time Grammy Award winner and drummer Paul Wertico, as well as Roscoe Beck and Larry Gray on bass and John Moulder on guitar, as well as others. People might remember Moulder from the concert he put on at the Sinnissippi Band Shell in summer 2001 when his guitar playing and arrangements had the audience speechless.
With a lineup of such well-seasoned musicians, Upper Palette should prove to be a musical mix-up of the best. The CD has been engineered and mastered by Daniel Leake, who has 23 Gold Records to his credit and has worked with the likes of Stevie Wonder, Janet Jackson and Sting. The exact date for the CD release party will be announced this month, so keep your eyes open.


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iPhone 4S Has Been Jailbroken, Hack Enables Siri on iPhone 4 148
from the making-changes dept.
We've improved Slashdot's video section; now you can view our video interviews, product close-ups and site visits with all the usual Slashdot options to comment, share, etc. No more walled garden! It's a work in progress -- we hope you'll check it out (Learn more about the recent updates).
I'm confused. Are we supposed to complain about having to jailbreak the 4S or whine about how dumb Siri is?
You shouldn't make these threads so complicated.
Since there's not much of a technical reason the iPhone 4 cannot have Siri, I think a big part of why it's only on the 4s (and not even the iPad 2) is to help Apple understand server load from the service before it goes live for 100 million+ people.
Are you kidding? It's a big part of the product differentiation for the iPhone 4s. They just launched the iPhone 4s and made a big deal out of the new voice assistant, Siri. They want people to buy the iPhone 4s for the new voice assistant, Siri. And you're wondering if the reason that they only make the new voice assistant, Siri, available for the iPhone 4s, the phone you'll have to buy if you you want the new voice assistant, is because they want to understand server load???
I wouldn't use it to book flights.
"Watson, I need a flight to Chicago."
"Booking your flight for Toronto; would you like an isle seat or WHAT IS LEG?"
that you have to preface your post with a disclaimer about the intensity of your dislike for a major corporation that doesn't know you exist? It speaks to something very sad about this community when you have pledge your intifada-like hatred for Apple as the intro to any post which is even tangentially related to Apple.
It would save time if you could just use a quick arm gesture and short phrase to make your allegiance clear. Maybe just shoot out your right arm straight at a 45 degree angle above the shoulder and say, "Heil, Google"--something like that.
The major difference between bonds and bond traders is that the bonds will eventually mature.


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More in Floors
Correctly calculating the placement of the first tile is the key to a successful tile installation.
Determine the longest run of the room. Measure from the outside wall to the center of this run and make a mark. Then go further down the outside wall and make another mark at the same distance from the wall.
Use these marks to snap a line the full length of the room. This line should be parallel to the outside wall. This line will be your reference mark to begin tiling. To set the line, spray it with hairspray.
Dry fit the tiles from the center line to the outside wall to determine where the first row of full tiles will be and to determine if and where you will have to cut tiles. Make a mark, transfer this same measurement down the wall and make another mark. Snap another chalk line using these two marks.


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Jon Stewart Points Out The Preposterous Debate Over Bill O'Reilly's 'Zones of Embellishment' - VIDEO
In the wake of the scandal surrounding Brian Williams' 'misremembering' an attack on a convoy he was traveling with during the Iraq War, media scrutiny has been re-focused on FOX News' Bill O'Reilly and allegations that he may have embellished his own experience during the Falklands War in 1982. However, as Jon Stewart point outs in a segment on last night's The Daily Show, going after O'Reilly for embellishing the truth might be a bit redundant:
Really?! We're going after O'Reilly for exaggerating being in a war zone? I don't know if you watch his program...misrepresenting 'the zone' he is in is kind of his hook. "You're in the no spin zone" are the words he utters right before throwing it to some jackass who disproves global warming by wandering around Boston by pointing at snow...You're basically putting in a tremendous amount of work to say, "The Emperor has no clothes," when the emperor has spent, like, twenty years going, "Look at my dick, I'm naked."
Watch Stewart dig into the debate and skewer just about everyone, AFTER THE JUMP...
Continue reading "Jon Stewart Points Out The Preposterous Debate Over Bill O'Reilly's 'Zones of Embellishment' - VIDEO " »


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Our special coverage of the closest race for years
Explore the personal stories of Syrian refugees in Lebanon
29 Mar 2011 08:13 GMT | Afghanistan, Chad
Maternal health is about more than just mothers and babies. Across the globe the very business of delivering life into the world is determined by power, politics and, all too frequently, poverty. In fact, maternal mortality is the greatest health inequality in the world. Ninety-nine per cent of women who die in childbirth are in the developing world and most of these deaths could be avoided if resources were invested to pay for adequate healthcare.Twenty million of the estimated 210 million women who become pregnant each year experience life-threatening complications and pregnancy is the biggest killer of women aged 15 to 19 in the developing world.A girl growing up in Chad today has about the same chance of dying in childbirth as she has of going to secondary school, while a 15-year-old girl in Afghanistan has a one-in-11 chance of dying in pregnancy or childbirth.Every year, 3.6 million babies die before they are a month old.We want to hear from you. What do you think the greatest concern is surrounding childbirth in your country? How do the aspirations and hopes of expectant parents vary from one country to another? What should be done to improve maternal health across the world? Tell us your stories relating to the world's greatest health inequality?
Source: Al Jazeera
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Critics say the UK's home secretary is using Muslims as a 'political punchbag' in a bid to shore up right-wing support.
Human Rights, Politics, Racism
2014 was the worst year in more than a decade for polio infections, with at least 306 new cases reported.
Polio, Asia, Pakistan
Recent court ruling finds police unit not guilty of rounding up and executing 42 Muslim men during 1987 communal riots.
Human Rights, India, Asia
Nigerians are flooding home for Saturday's vote, but fears of violence grow amid the too-close-to-call election.
Nigeria Decides, Nigeria, Africa
It is a source of sustenance but also of tension and new political realities are now increasing uncertainty.
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A media post-mortem into the UK's surveillance report. Plus, why five years on in Argentina the media looks so familiar.
Media, Edward Snowden, Security
The untold story of how the Lebanese community overcame the odds and found their place in multicultural Australia.
Asia Pacific, Australia, Lebanon
How to keep a telenovela profitable and demanding sponsors happy while inspiring the production team to stay passionate.
Arts & Culture, Mexico, Latin America


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Can the rough draft sometimes be better than a final copy?
"The band decides to test run a new set of songs live, in performance before ever entering a music studio. Teaming up with local filmmakers in the process, the results were raw and roughhewn: a cinéma-vérité of songwriting & its initial muse in the earliest stages, before the producer or studio can sculpt it different. Shot in stark black and white & captured in 1 day of full-shoot recording on a particularly hazy afternoon in a particularly San Francisco garage, the footage asks the question: will this be Growwler's rough draft or final copy?" (Text by Pete Gast, Stentorian Sounds 2013)
Filmed and recorded by Ben Ambrogi & Joe Zolnoski on April 18 of 2013.
For more Growwler, please go here: Www.growwler.coM
For more of the video directors and crew, go here:
Josef Zolnoski: josefzolnoski.com
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Content Rating and Approval Process Details
View Single Post
Archived Post
Lt. Commander
Join Date: Dec 2007
Posts: 120
# 85
Originally Posted by
Although it is a sentiment I can appreciate, I don't think that, for example, movie critics are required to have directed or produced a movie before they write their reviews. I'm sure that there are some things in UGC missions that are bugged, which is no fault of the author - but someone playing the mission will expect the author to have played through and changed buggy things. That might not be the main issue, however; if the issue is people rating things based on content and story, I don't have a problem with anyone ripping missions apart.
I agree with you about not having to have created missions in order to review content. That would be silly. I also agree with you about ripping a mission apart. That is up to the individual reviewer and how they want to handle it. I try to submit a review that may help the author improve the mission. In the end I feel that will improve UGC missions and make the game better for everyone.


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Naylor Creek plants arrive
Here they are, mostly golden and shining like new money.
Teatime with some shoots and fat eyes and then open leaves
Stained Glass replacement just in case
Forbidden Fruit, sport of Orange Marmalade....tet? I forget
Funky Monkey
Paradise Island x 2...can you tell I LIKE IT?
Orange Marmalade...much bigger than I expected it to be
They look great! Naylor is a great source and I'm sure they will all be lovely in your garden. I have Funky Monkey and it is looking pretty darn nice this year and definitely "funky" LOL! If you want to see a pic of mine - look at my thread for "What looks good today Apr 26". The thumbnail is clickable.
By the time I got these in the pots today, the Stained Glass had unfurled two leaves completely. No longer just shoots. Here it is with my older Stained Glass which never went dormant, has yet to grow any new leaves this spring. I placed the two pots next to each other, did not want to pot them together, which I'd have done if I knew it was cool to do so.
Teatime with a green teapot to help me remember where it is.
Stained Glass #1 left and new guy #2 right
Funky Monkey
Forbidden Fruit
Orange Marmalade
and.....(drum roll)....PARADISE ISLAND x2
Sure hope you like them. I feel happy as a clam.
BKay, a lot of time potting, and all the time looking for new pots, more potting soil, insecticide, slug pellets, you name it. Then pictures, and I try to "tag" them and have to upload them to Flickr. (I had troubles with Photobucket so I stopped loading there, just keep the account for viewing others' albums). It takes a lot of time to identify each photo, else I'd wind up with a lot of PHOTO NOIDS.
However, I think Paradise Island is one that I could ID from further than 10 feet! I'll be taking a fun trip to a cheap store to look for small figures that can be used in hosta pots to help jog my memory about its name. I saw some ceramic stakes with mushrooms all colors on top, that would be nice size (about 2 inch figure on top of a 6-8 inch stake) for a pot of hosta, but no good to have mushrooms. And NO SNAILS up on top either thank you!
Maybe some nice size rocks for names? But in pots, the rocks get covered up.
Sometimes I tuck one of those plastic markers just inside the edge so it sits just above soil level, but below the edge of the pot. When I forget a name I can look in there, but it doesn't stick up above the level of the pot.
If you have so much room around your hosta in the pot, you might have it in too large a pot. Just a suggestion.
Go to a Staples kind of store and get a label printer. I leave my names where I can see them and rearrange them for photos so I do not have to id later. When I do the hosta walk I can call them out by name. Have a great day all, Paula
Lovely pictures there in their jars.
Babka, I was going by the size of the roots, which were extensive and needed room to spread out. Seems I read that could require a larger pot.
The teapot is barely in the pot, it will be coming out and I'll use it for watering with liquid fertilizer. I have some fish emulsion.
Folks keep saying how hosta don't like to be moved or repotted it could set them back. So I'm giving them a bit of space, but not really too too much. I have big holes drilled in all the plastic pots, usually 3 or 4, and then a lot of styrafoam peanuts beneath the media, now I have all that lovely bark and MiracleGro which will help them drain. So if there is a larger margin in the pot, perhaps they won't suffer. The small plants are all in small pots.


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- Cooking Time: 0
- Servings: 1
- Preparation Time: 5
- 3 ounces (1/4 cup, plus 2 tablespoons) canned tropical fruit salad in natural juices (from a 15-ounce can)*
- 3 ounces (1/4 cup, plus 2 tablespoons) white cranberry juice drink
- 2 ounces (1/4 cup) ginger ale
- Ice
- Canned pineapple wedge for garnish (optional)
- Equipment:
- Martini shaker or pint glass
- Shaking tin
- Cocktail strainer
- Do ahead: In blender, purée entire can of tropical fruit salad until smooth. Set tropical fruit purée into a separate bowl/pitcher. Rinse blender bowl and return to base.
- To make one drink: In martini shaker, add 3 ounces tropical fruit purée and white cranberry juice. Fill martini shaker with ice, and shake vigorously 12 times. Pour into pint glass; top with ginger ale. Stir gently. Garnish with pineapple wedge.
- Tip: Reduce 1 1/2 ounces of white cranberry juice and add 1 1/2 ounces light or spiced rum to turn this into an adult-only treat.
- *Note: One, 15-ounce can of tropical fruit salad in natural juices makes 5 drinks.
- Serving glass: 16-ounce pint glass
- Servings: 1
- Nutritional Information Per Serving: Calories 110; Total fat 0g; Saturated fat 0g; Cholesterol 0mg; Sodium 25mg; Carbohydrate 28g; Fiber 1g; Protein 0g; Vitamin A 0%DV**; Vitamin C 70%DV; Calcium 2%DV; Iron 4%DV **Daily Value
NotesAdd some sparkle to your afternoon. This fruity pick-me-up – made with vitamin-C rich fruit – is refreshing.
MetroCooking DC 2013
Cooking with THE HOLMES SISTERS Volume 1 "As seen on 3ABN"
Laissez Les Bons Temps Rouler! Delicious Recipes For Any Get Together or PartySee More
Molten Chocolate Walnut Cupcakes
Rabiole Soup (white turnip)
Stamp you passport ~> Portuguese HamburgersSee More


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Acquia Cloud is looking for senior software engineers to help build its cloud platform as a service on distributed systems and cluster computing technologies. Acquia Cloud is a enterprise grade Platform as a Service (PaaS) hosted entirely on Amazon Web Services. We are one of3/19/2015 Boston, MA Save This Job
Job Description At CloudFlare, we have our eyes set on an ambitious goal -- to build a better web. We believe that with our talented team, smart technology and engaged users we can solve some of the biggest problems on the web. We already serve more web traffic than Twitter,2/7/2015 San Francisco, CA Save This Job
We are seeking a Sr. Software Engineer to help build out TubeMogul's next generation of elegant and responsive web interfaces. The role will require hands-on experience across the stack- from MySQL query optimization to cross-browser responsive HTML/CSS. Candidates will be2/1/2015 Emeryville, CA Save This Job
DESCRIPTION We are looking for a Sales Engineer/Product Specialist to help support our growing business development team and customer pipeline. In addition the Sales Engineer will play an active role in contributing to the overall success of projects on behalf of our global2/25/2015 Palo Alto, CA Save This Job
Come join the #1 Fastest Growing Company in the Bay Area! With a whopping 4,700% revenue growth and 23 billion monthly page views, it is no wonder why we are ranked among the Top 40 Forbes America?s Most Promising Companies. ShareThis offers the most innovative sharing platform2/22/2015 Palo Alto, CA Save This Job
Job Description Position Summary: We have an opportunity that will challenge you, push your engineering boundaries, and grow your skills to their highest levels. Our solutions are innovative, obsessively user friendly, and built upon a flexible and robust architecture that2/1/2015 Fargo, ND Save This Job
The Systems Engineer?s role is to ensure the stability, integrity and efficient operation of the information systems that support core organizational functions. This is achieved by monitoring, maintaining, supporting and optimizing all hardware, storage, networking, operating2/1/2015 Detroit, MI Save This Job
SAN MATEO, CA JOB DESCRIPTION We are looking for a talented software engineer who thrives on developing scalable, reliable, secure platform that processes millions of dollars of transactions per day through various billing mechanisms including direct carrier billing, credit card2/22/2015 San Mateo, CA Save This Job
RockYou, a leading social gaming company is seeking a Senior Software Engineer to be part of the engineering team and help design and build the next generation features for social games. Responsibilities: Be capable of working independently, designing and delivering2/21/2015 San Francisco, CA Save This Job
As a software engineer on the Site Reliability Engineering team, you will be responsible for building out new automation capabilities to measure and validate the AppNexus platform. You will primarily develop code in PHP or Python and leverage open source testing technologies such2/12/2015 New York, NY Save This Job
Emeryville, CA We are seeking a Software Engineer to help build out TubeMogul's next generation of elegant and responsive web interfaces. The role will require hands-on experience across the stack- from MySQL query optimization to cross-browser responsive HTML/CSS. Candidates3/3/2015 Emeryville, CA Save This Job
Job Type: Full Time Location: San Mateo, CA Department: Business Services Since 2003, RingCentral has been breaking down the communication barriers created by complex on-premise hardware. RingCentral delivers cloud business communications solutions that frees people to work the2/21/2015 San Mateo, CA Save This Job
RockYou (www.rockyou.com) serves billions of ad impressions on major social networks and is looking for a Staff Software Engineer - Analytics to join the team. Responsibilities: Design and implement major systems within the analytics system with little or no management2/21/2015 San Francisco, CA Save This Job
Job Descriptions Senior Software Engineer focusing on development of .NET and Java based applications and SDKs. The role will require the design, development and testing of various software components for RFID readers. As a member of a multi-functional Software Development team2/16/2015 Morgan Hill, CA Save This Job
Thumb is a fast-growing, venture-backed, NYC-based mobile service that helps users get and give opinions in realtime. We?ve built an addicting mobile application that?s growing rapidly, providing millions of rapid responses per day to users asking all manner of questions on2/1/2015 New York, NY Save This Job
Location: Philadelphia, PA Department: Engineering Type: Full Time Min. Experience: Mid Level We are looking for a Systems Engineer to join our incredibly successful team. Are you self-motivated, energetic, and have excellent interpersonal skills? Do you want to join a1/25/2015 Philadelphia, PA Save This Job
New York, NY Design & implement computer algorithms; analyze computer hardware & software problems by conducting technical & logical analyses; apply theoretical expertise & innovation to create new technologies; develop mathematical models for computers & implementation of3/27/2015 New York, NY Save This Job
RESPONSIBILITIES: Develop and maintain dynamic, high traffic web applications and high performance internal products. Estimate work, plan implementation, develop quality code on time, and compile technical documentation. Provide support to users with technical problems. Keep3/20/2015 Irvine, CA Save This Job