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Cell Reports, Volume 2, Issue 5, 1272-1285, 01 November 2012
Proliferative and Survival Effects of PUMA Promote Angiogenesis
Fan Zhang1, Yang Li1, Zhongshu Tang1, 2, Anil Kumar1, Chunsik Lee1, Liping Zhang3, Chaoyong Zhu4, Anne Klotzsche-von Ameln5, Bin Wang6, Zhiqin Gao7, Shizhuang Zhang8, Harald F. Langer9, Xu Hou10, Lasse Jensen11, 12, Wenxin Ma1, Wai Wong1, Triantafyllos Chavakis5, Yizhi Liu2, Yihai Cao11, 12 and Xuri Li1, 2, ,
1 National Eye Institute, National Institutes of Health, Rockville, MD 20852, USA
2 State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, P.R. China
3 National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD 20892, USA
4 School of Public Health, NanTong University, 9 Seyuan Road, Nantong 226019, P.R. China
5 Division of Vascular Inflammation, Diabetes and Kidney, Department of Medicine, and Institute of Physiology, University Dresden, Dresden 0351 463-0, Germany
6 Medical Imaging Institute, Shandong Province Characteristical Key Subject, Medical Imaging and Nuclear Medicine, Binzhou Medical University, Yantai, 264003 P.R. China
7 Department of Cell Biology, Weifang Medical University, Weifang, 261053, P.R. China
8 Department of Radiology, Medical Imaging Centre of the Affiliated Hospital, Weifang Medical University, Weifang, 261053, P.R. China
9 Department of Cardiovascular Medicine, University of Tübingen, Tübingen 72074, Germany
10 Department of Ophthalmology, Eye Institute of Chinese PLA, Xijing Hospital, Fourth Military Medical University, Xi`an, Shaanxi 710032, China
11 Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, 171 77 Stockholm, Sweden
12 Department of Medicine and Health Sciences, Linköping University, 581 83 Linköping, Sweden
Graphical Abstract
► PUMA is abundantly expressed in vascular and microglia cells ► PUMA promotes vascular/microglia cell proliferation/survival and angiogenesis ► Puma deficiency reduces angiogenesis and microglia density ► PUMA regulates autophagy, Erk activation, and intracellular calcium level
- The p53 upregulated modulator of apoptosis (PUMA) is known as an essential apoptosis inducer. Here, we report the seemingly paradoxical finding that PUMA is a proangiogenic factor critically required for the proliferation and survival of vascular and microglia cells. Strikingly, Puma deficiency by genetic deletion or small hairpin RNA knockdown inhibited developmental and pathological angiogenesis and reduced microglia numbers in vivo, whereas Puma gene delivery increased angiogenesis and cell survival. Mechanistically, we revealed that PUMA plays a critical role in regulating autophagy by modulating Erk activation and intracellular calcium level. Our findings revealed an unexpected function of PUMA in promoting angiogenesis and warrant more careful investigations into the therapeutic potential of PUMA in treating cancer and degenerative diseases.
The p53 upregulated modulator of apoptosis (PUMA) is a member of the BH3-only protein family and has been known as an essential apoptosis inducer for many years (Chipuk et al., 2005; Vousden, 2005; Yu and Zhang, 2003). The apoptosis-inducing activity of PUMA has been shown in different cell types, such as mouse embryonic fibroblasts, developing neurons, lymphocytes, tumor cells, and intestinal progenitor/stem cells (Michalak et al., 2010; Villunger et al., 2003). Due to its potent apoptotic activity, PUMA has received considerable research interest and has been considered as an important therapeutic candidate gene for the prevention and treatment of cancer and degenerative diseases (Ito et al., 2005; Yu, 2009).
Despite many years of intensive studies on PUMA, outstanding questions remain. Numerous studies have shown that enhanced PUMA expression is not always linked to apoptosis. Interaction of human umbilical vein endothelial cells (HUVECs) with monocytes increased PUMA protein level in the former (Gawad et al., 2009), yet the elevated PUMA protein level did not result in apoptosis of the cells (Gawad et al., 2009). In brain ischemia, PUMA expression was markedly upregulated (Kuroki et al., 2009). However, apoptosis was not increased, and the brain stroke volume was unchanged in the Puma-deficient mice (Kuroki et al., 2009). The biological consequences of such increased PUMA expression remain thus far unclear, indicating potentially unknown functions of PUMA other than induction of apoptosis. In addition, studies have shown that the proapoptotic action of PUMA may be cell specific. For example, although Puma deficiency reduced apoptosis in intestinal progenitor/stem cells, it did not affect apoptosis of endothelial cells (Qiu et al., 2008). Moreover, PUMA is highly expressed in vascular and microglia cells (Table S1. Puma Is Highly Expressed in Myeloid and Vascular Cells, as well as in Other Human Tissues and Cells, Related to Figure 1 and Table S2. Puma Is Highly Expressed in Macrophages and Microglia, as well as in Other Mouse Tissues and Cells, Related to Figure 1), yet it has not been shown to induce apoptosis in these cells thus far. Instead, little is known about the function of PUMA in vascular and microglia cells. Of note, PUMA was recently reported to be required for tumor development, whereas Noxa, another proapoptotic gene of the BH3-only protein family, inhibited tumorigenesis (Labi et al., 2010; Michalak et al., 2010; Qiu et al., 2011), demonstrating a distinct proliferative effect of PUMA compared with the other proapoptotic genes. Together, accumulating data have indicated a potential unknown function of PUMA not related to apoptosis, at least in certain types of cells.
Microglia are resident macrophages of the CNS. Although the immune function of microglia has been widely studied, their role in angiogenesis is not well understood. Some studies reported an angiogenic effect of microglia (Checchin et al., 2006; Liu et al., 2005; Yamaguchi et al., 2011), whereas others reported an antiangiogenic effect (Stefater Iii et al., 2011). Interestingly, in the developing mouse retina, the microglia density increases during the first postnatal week and decreases gradually (Santos et al., 2008). The appearance of retinal microglia precedes and overlaps with the formation of retinal blood vessels, indicating a potential role of microglia in retinal angiogenesis. Further studies are needed to better understand the function of microglia in blood vessel growth.
In the present study, we investigated the effect of PUMA on vascular and microglia cells. We provide evidence that PUMA is an angiogenic factor critically required for the proliferation and survival of vascular and microglia cells by modulating autophagy. At the molecular level, we revealed that PUMA plays a crucial role in regulating Erk activation and intracellular calcium level. Our data revealed an unexpected function of PUMA as an angiogenic factor by promoting survival and proliferation, which contradicts its known role as an essential apoptosis inducer.
PUMA Is Expressed by Retinal Microglia and Blood Vessels
Although PUMA has been intensively studied for many years, its role in the retina remains unexplored. Therefore, we investigated the retinal expression pattern of PUMA. Real-time PCR displayed a postnatal increase in PUMA expression and a subsequent decrease after postnatal day 18 (P18) (Figure 1A). This was confirmed at the protein level by western blot (Figure 1B). Immunofluorescence staining detected abundant PUMA expression in blood vessels (Figure 1C) and CD11b+ microglia in the developing mouse retinae (Figure 1D). PUMA expression was also identified in primary retinal microglia isolated from neonatal mice (Figure 1D). Notably, PUMA expression levels in the developing mouse retinae coincided with the time window of retinal blood vessel development, which occurs mainly within the first postnatal 2 weeks. Moreover, the retinal PUMA expression levels paralleled the time window of the emergence of retinal microglia cells, the number of which increases in the retina during the first postnatal week and decreases gradually thereafter (Santos et al., 2008). In neonatal mice, the retinal microglia cells are mainly present in the retinal ganglion cell (RGC) layer and the inner nuclear layer (INL) (Santos et al., 2008), where PUMA expression is abundant (Figure 1C). Thus, even though the changes in retinal PUMA expression (Figures 1A and 1B) may reflect changes in PUMA expression in retinal neurons, the spatiotemporal correlation of the expression level and distribution of PUMA with retinal blood vessel formation and the appearance and location of retinal microglia cells suggest a potential effect of PUMA on retinal microglia and blood vessels.
Puma Deficiency Decreased Microglia and Blood Vessel Density in the Retina
We subsequently investigated the role of PUMA in the retina using Puma-deficient mice. Because PUMA is known as an essential apoptosis inducer (Chipuk et al., 2005; Vousden, 2005; Yu and Zhang, 2003), we hypothesized that Puma deficiency might lead to decrease apoptosis in the retina. Surprisingly, we found a marked decrease of retinal microglia numbers in the Puma-deficient mice (Figures 1E, 1F, S1A, and S1B). Importantly, loss of one Puma allele, i.e., loss of half of its normal expression level (Figure S1C), decreased retinal microglia density at different time points (Figures S1D–S1F). Puma deficiency also decreased retinal blood vessel branch points, vessel areas, and diameter (Figures 1F, S1G, and S1H).
To further verify these findings, we intercrossed Puma-deficient mice with Cx3cr1GFP/+ transgenic mice, in which microglia are GFP labeled (GFP expression under the control of a Cx3cr1 promoter) with a normal density and morphology (Davalos et al., 2005). We found that the retinal microglia and blood vessel densities decreased in the Puma-deficient Cx3cr1GFP/+ mice at different time points (Figures 1G and S1I). The decreased retinal blood vessel branch points, vessel areas, and diameters were also observed in the retinae of 6-month-old Puma-deficient mice (Figure 1H). We next investigated microglia and blood vessel density in the brains of Puma-deficient mice to verify whether the effect of PUMA was retina specific. We found reduced microglia (Figure S1J) and blood vessel density in the brains of Puma-deficient mice as well (Figure S1K).
Puma Knockdown by Small Hairpin RNA Reduced Microglia and Blood Vessel Density in the Retina
The observation of decreased microglia and blood vessel density in the Puma-deficient retina and brain was unexpected, given the known apoptotic activity of PUMA (Chipuk et al., 2005; Vousden, 2005; Yu and Zhang, 2003). To substantiate our findings, we performed a loss-of-function assay using Puma small hairpin RNA (shRNA). Intravitreal injection of mouse Puma shRNA at P1 decreased PUMA expression level by about 60% in the retinae (Figures S2A and S2B) and decreased retinal microglia number, blood vessel branch points, and vessel areas at P6 (Figure 2A).
Puma Deficiency Did Not Affect Retinal Astrocytes or Decrease Retinal Thickness
Because retinal astrocytes are known to contribute to retinal angiogenesis, we investigated whether Puma deficiency affected the retinal astrocyte network and found no significant difference in astrocyte area or morphology between wild-type and Puma-deficient retinae (Figure S2C). We further assessed vascular endothelial growth factor (VEGF) expression levels in Puma-deficient and wild-type retinal astrocytes and found a slightly higher VEGF protein level in Puma-deficient astrocytes (Figure S2D). Similarly, real-time PCR and western blot showed that VEGF expression level was somewhat higher in the Puma-deficient retinae (Figures S2E and S2F). Thus, the vascular defects in the Puma-deficient retinae were unlikely due to a potential astrocyte or VEGF defect.
Loss of retinal neurons can subsequently lead to loss of retinal blood vessels (Zheng et al., 2007). We next investigated whether Puma deficiency resulted in retinal neuronal loss, which could in turn explain the angiogenesis defect. We measured the thickness of the retinal layers where the retinal blood vessels exist, i.e., the RGC layer, the inner plexiform layer (IPL), and the INL. We found no significant difference in the thickness of the retinal layers before birth at E18 (Figure S2G). After birth, in Puma-deficient mice, the thicknesses of the RGC, IPL, INL, and the whole retina were increased (Figures S2H–S2J). There was no significant difference in the thickness of the other retinal layers in Puma-deficient mice (data not shown). Moreover, PUMA overexpression in the retinal ganglion cell line (RGC5) markedly induced apoptosis (Figures S2K–S2M), consistent with the in vivo observation that Puma deficiency increased thickness of the retinal ganglion cell layer (Figures S2H–S2J). In summary, Puma deficiency did not lead to retinal neuronal loss.
To address whether the effect of PUMA on retinal microglia and blood vessels was a common event of the BH3-only protein family genes, we utilized Noxa-deficient mice. Like PUMA, Noxa is also a p53-upregulated proapoptotic gene of the BH3-only protein family. However, no significant microglia or vascular defects were observed in Noxa-deficient mice (Figure S2N), demonstrating a functional uniqueness of PUMA.
Retina Microglia Cells Are Required for Retinal Angiogenesis
Microglia cells are present in the mouse retina as early as at embryonic day 11.5 (E11.5) (Santos et al., 2008), before the mouse retinal vasculature starts to develop at postnatal day 1 (P1). Because the observed retinal microglia cell defect precedes the retinal blood vessel defect in the Puma-deficient mice, we hypothesized that microglia loss in the Puma-deficient retinae might relate to the vascular defect. To test this, we depleted the mouse retinal microglia at P1 (a time point when retinal angiogenesis just begins) by intravitreal and intraperitoneal injection of clodronate liposomes, which reduced retinal microglia numbers by about 50% (Figure 2B). Microglia depletion decreased retinal blood vessel branch points and vessel areas (Figure 2B), demonstrating that retinal microglia are required for retinal angiogenesis. Consistently, real-time PCR showed that isolated primary retinal microglia expressed numerous angiogenic factors (Figure 2C). Importantly, Puma deficiency by genetic deletion or shRNA knockdown markedly decreased the expression of many proangiogenic genes in primary retinal microglia in vitro and in the retina in vivo (Figures 2D and 2E). Vice versa, overexpression of PUMA upregulated the expression of some angiogenic genes (Figure 2F). Western blot confirmed the downregulation of some important angiogenic factors, such as fibroblast growth factor 2 (FGF2), IL6, and CCL2, in Puma-deficient retinae (Figures 2G–2I). Together, these data demonstrate that retinal microglia contribute to retinal angiogenesis by producing important angiogenic factors.
Impaired Angiogenic Response in Puma-Deficient Aortic Ring and Cornea
We subsequently used an aortic ring and a cornea pocket assay to investigate whether Puma deficiency affected angiogenesis in other tissues. In the aortic ring assay, Puma deficiency nearly completely abolished VEGF- and FGF2-induced angiogenesis (Figures 3A and 3B). Immunofluorescence staining revealed less CD11b+ cells in Puma-deficient aortic rings (Figure 3C). In the cornea pocket assay, Puma deficiency impaired VEGF-induced angiogenesis (Figures 3D and S3A–S3C). Immunofluorescence staining revealed fewer CD11b+ cells in Puma-deficient corneas (Figure 3E). Thus, Puma deficiency impaired angiogenesis in other tissues than the retina.
Puma Deficiency Inhibited Choroidal Neovascularization
We next tested whether Puma deficiency affected choroidal neovascularization (CNV) using Puma-deficient mice and Puma shRNA in a laser-induced CNV model. Puma deficiency decreased CNV area by more than 50% (Figure 3F). Moreover, intravitreous injection of Puma shRNA, which decreased PUMA expression by about 60% in the retinae and choroids (Figure S3D), inhibited CNV formation (Figure 3G) and decreased the number of CNV-associated Iba1+ microglia (Figure 3G). Indeed, real-time PCR displayed decreased expression of different microglia markers, including Iba1 (Figure 3H), CD11b, and CD68 (Figure S3E) in the Puma shRNA-treated choroids and retinae. Consistent with the reduced CNV areas by Puma shRNA treatment, the expression of many important angiogenic genes markedly decreased in the Puma shRNA-treated choroids and retinae (Figure S3F).
PUMA Is Required for Vascular and Microglia Cell Proliferation and Survival
To investigate the cellular mechanisms underlying the effect of PUMA, we explored whether PUMA affected vascular and microglia cell proliferation and survival. In cultured primary retinal microglia, brain endothelial cells (ECs), and aortic artery smooth muscle cells (SMCs), Puma deficiency decreased cell proliferation and survival under different conditions (Figures 4A–4C). Consistently, Puma knockdown by shRNA, which decreased PUMA expression in primary microglia by about 40% (Figures S4A and S4B), decreased viability of primary retinal and brain microglia (Figures 4D and S4C). On the other hand, PUMA overexpression increased survival of primary retinal microglia cells (Figures 4E, 4F, and S4D) and proliferation of rat retinal vascular endothelial cells (TR-iBRB) and HUVECs (Figures 4G, 4H, S4E, and S4F). PUMA overexpression did not induce, but instead inhibited, apoptosis in the rat retinal vascular endothelial cells (TR-iBRB, Figures S4G and S4H).
We next engaged several more direct methods than the MTT assay to further verify the proliferative and survival effect of PUMA, such as the BrdU assay, the trypan blue exclusion assay, and the LIVE/DEAD viability/cytotoxicity assay. Using the BrdU assay, we found that overexpression of PUMA increased cell proliferation in the primary microglia cells (Figures 4I and S4I), TR-iBRB, and HUVECs at different time points (Figures 4J, 4K, S4E, and S4F), whereas PUMA deficiency by shRNA knockdown inhibited proliferation of primary microglia (Figures 4L and S4J). To investigate the survival effect of PUMA, we treated the cells with mitomycin C to suppress cell proliferation, and performed a trypan blue exclusion assay and a LIVE/DEAD viability/cytotoxicity assay. Using these two methods, we found that overexpression of PUMA increased survival of primary microglia (Figures 4M and S4I), whereas Puma shRNA treatment decreased their survival (Figures 4N, 4O, and S4J). Thus, apart from the MTT assay, several additional assays demonstrated a proliferative and survival effect of PUMA on microglia and vascular cells. Furthermore, Puma deficiency impaired retinal microglia migration induced by ATP, CCL2, and CCL3 (Figure 5A).
Puma Deficiency Impairs Erk Activation and M-CSFR Expression
We subsequently investigated the molecular mechanisms underlying the actions of PUMA. Puma deficiency markedly attenuated Erk activation under different conditions in primary brain vascular endothelial (Figure 5B) and retinal microglia cells (Figure 5C). Consistently, Puma knockdown by shRNA inhibited Erk activation in primary microglia (Figures 5D, S4A, and S4B). In addition, the expression level of macrophage colony stimulating factor receptor (M-CSFR), a factor mainly expressed by microglia/macrophages and critical for angiogenesis (Kubota et al., 2009), considerably decreased in Puma-deficient microglia and retinae (Figures 5E and 5F). This observation is consistent with the finding of less microglia in Puma-deficient retinae. Moreover, the expression levels of M-CSFR in cultured primary microglia in vitro and in mouse retinae in vivo decreased after Puma shRNA treatment (Figures 5G, 5H, S2A, and S2B). Importantly, Puma gene delivery by in vivo transfection of PUMA-expressing DNA plasmids into P1 Puma-deficient mouse retinae increased retinal microglia numbers (Figures 5I, 5J, and S5A–S5C), retinal vascular branch points and vascular area (Figures 5K and 5L), and restored M-CSFR expression and Erk activation at P6 (Figure 5M). Thus, PUMA is critically required for Erk activation and M-CSFR expression, both of which are important for angiogenesis and microglia proliferation, survival, and migration (Liva et al., 1999; Otero et al., 2009; Reddy et al., 2009; Sasaki et al., 2000).
PUMA Is Required for Vascular and Microglia Cell Autophagy
Our finding that PUMA is required for vascular and microglia cell proliferation and survival was unexpected, as it contradicted the common belief that PUMA is an essential apoptosis inducer. To understand this paradox, we investigated whether PUMA affected autophagy, a critical regulatory process of cell survival and proliferation, under conditions of metabolic stress (Rabinowitz and White, 2010). The intensity of the LC3II band (16 kDa) was used as a marker for autophagy using western blots. Puma deficiency markedly impaired autophagy under various conditions in primary retinal microglia (Figure 6A), brain vascular ECs (Figure 6B) and aortic artery SMCs (Figure 6C), as demonstrated by the disappearance or reduced intensity of the 16 kDa LC3II band (Figures 6A–6C). Consistently, Puma shRNA treatment markedly inhibited autophagy in the RAW264.7 macrophage cells (Figures 6D and S6A), whereas overexpression of PUMA increased autophagy in RAW264.7 cells and primary retinal microglia (Figures 6E, 6F, S6B, and S6C). Moreover, immunofluorescence staining showed that overexpression of PUMA in primary retinal microglia increased autophagy as demonstrated by the increased LC3+ dots (Figures 6G [arrows] and S6C), whereas Puma deficiency decreased autophagy in primary brain ECs (less LC3+ dots) (Figure 6H, arrows).
PUMA-Induced Autophagy Requires Erk Activation
Because Puma deficiency attenuated Erk activation (Figures 5B–5D), we hypothesized that PUMA-induced autophagy might require Erk activation and tested this hypothesis. Treatment with an Erk1/2 inhibitor U0126 inhibited Erk activation and PUMA-induced autophagy in primary microglia (Figures 6I and S6C) and TR-iBRB (Figures 6J and S4E). Consistently, immunofluorescence staining showed that while PUMA overexpression induced autophagy in the TR-iBRB cells (Figure 6K, middle panel, arrows showing the increased LC3+ dots), U0126 treatment abolished PUMA-induced autophagy (Figure 6K, right panel, decreased LC3+ dots). Moreover, western blot showed that U0126 treatment inhibited Erk activation and autophagy (reduced intensity of the LC3II band) in the aortic SMCs (Figure 6L). We next investigated whether Erk activation was responsible for the greater survival rate of the wild-type microglia and vascular cells. We found that U0126 and A6355 (another Erk inhibitor) treatment completely abolished the increased survival rate of the wild-type microglia and vascular cells (Figures 6M–6O). Thus, our data demonstrated that Erk activation was required for PUMA-induced autophagy and survival of vascular and microglia cells.
PUMA Deficiency Decreased Vascular and Microglia Cell Cytoplasmic Calcium Levels
It is known that in fibroblasts PUMA, but not Noxa, increases cytosolic calcium levels (Shibue et al., 2006). Importantly, cytoplasmic calcium plays a critical role in inducing autophagy (Høyer-Hansen et al., 2007; Swerdlow and Distelhorst, 2007) and Erk activation (Agell et al., 2002; Schmitt et al., 2004). We therefore studied whether PUMA affected intracellular calcium levels in vascular and microglia cells. Using fluorescent Fluo-4 acetoxymethyl (Fluo-4AM) as an indicator of cytosolic calcium, we found that Puma deficiency reduced cytosolic calcium levels under a steady-state condition and upon ATP or mechanical stimulation in primary retinal microglia (Figure 7A), primary vascular ECs (Figure 7B), and primary vascular SMCs (Figure 7C). Consistently, Puma knockdown by RFP-Puma shRNA decreased cytosolic calcium levels in primary retinal microglia in the RFP-Puma shRNA expressing cells (Figure 7D, arrows in the right panel). These data demonstrated that PUMA plays a critical role in regulating cytosolic calcium levels, which is essential in modulating Erk activation and autophagy.
Our findings that PUMA is critically required for the survival and proliferation of vascular and microglia cells and plays important roles in angiogenesis are highly unexpected, given that PUMA has been considered as an essential apoptosis inducer for more than 10 years (Yu and Zhang, 2008). We further revealed that the survival/proliferative effect of PUMA was achieved by induction of autophagy, a critical mechanism for cells to respond to metabolic stress in order to survive, proliferate, and migrate (Fan et al., 2010). Mechanistically, we revealed that PUMA plays a critical role in regulating Erk activation and intracellular calcium level. Our data thus revealed an unexpected function of PUMA as a survival, proliferative, and angiogenic factor that contradicts the known apoptosis-inducing role of PUMA.
Little was known about the function of PUMA in vascular and microglia cells, even though the proapoptotic effect of PUMA on other types of cells has been studied intensively. Indeed, although Puma deficiency in mice decreased apoptosis in fibroblasts and lymphocytes (Villunger et al., 2003), the same apoptotic effect has not been observed on vascular or microglia cells. In this study, we found that PUMA is abundantly expressed in vascular and microglia cells. In vitro, Puma deficiency impaired proliferation and survival of cultured vascular and microglia cells, and nearly completely abolished the angiogenic response of mouse aortic rings. In vivo, Puma deficiency by genetic deletion or shRNA treatment decreased blood vessel and microglia density in retina and brain, and suppressed angiogenesis in different animal models. Consistently, Puma gene delivery promoted cell proliferation and survival in vitro, and increased blood vessel and microglia density in vivo. Interestingly, PUMA was recently reported to be critically required for tumorigenesis (Michalak et al., 2010), whereas other apoptotic genes, such as p53 and Noxa, inhibited tumorigenesis (Michalak et al., 2010). Although a different mechanism underlying the tumorigenic activity of PUMA was suggested, it nevertheless indicated a unique growth-promoting function of PUMA, at least under certain conditions. In addition, while retinal microglia density and angiogenesis were reduced in Puma-deficient mice, the retinal astrocytes appeared to be normal, demonstrating a cell-specific effect of PUMA. Indeed, other investigators have also reported a cell-specific effect of PUMA (Qiu et al., 2008).
Another important finding in this study is the critical role of PUMA in inducing vascular and microglia cell autophagy by regulating Erk activation and cytosolic calcium levels (Figure 7E). Autophagy is a homeostatic cellular recycling mechanism essential for cell survival, proliferation, and migration (Fan et al., 2010; Rabinowitz and White, 2010). It is especially crucial when the cells are under metabolic stress, such as hypoxia, even though uncontrolled autophagy can eventually lead to cell death. Autophagy has been shown to be critically required for macrophage migration and T cell proliferation (Kadandale et al., 2010). In this study, loss of PUMA by gene deletion or shRNA markedly inhibited autophagy and impaired Erk activation in microglia, vascular ECs, and SMCs, whereas Puma gene delivery increased autophagy and Erk activation. Calcium signaling plays a critical role in Erk activation (Agell et al., 2002; Schmitt et al., 2004) and autophagy induction (Høyer-Hansen et al., 2007; Swerdlow and Distelhorst, 2007). Because shRNA-mediated loss of PUMA markedly decreased cytosolic calcium levels in microglia, the effect of PUMA on Erk activation and autophagy may be mediated by its regulatory effect on calcium signaling.
The previously unrecognized angiogenic effect of PUMA is surprising, because no other BH3-only protein family member has been shown to display such a function. It is reported that microglia play crucial roles in angiogenesis, at least in part, by producing potent angiogenic factors (Checchin et al., 2006; Liu et al., 2005; Yamaguchi et al., 2011). In this study, we found that the expression levels of many important angiogenic factors were significantly reduced. Thus, apart from its direct effect on vascular cells, PUMA plays an important role in angiogenesis also by ensuring adequate microglia density and sufficient amount of angiogenic factors.
Our finding that PUMA is a survival, proliferative, and angiogenic factor may have important therapeutic implications. Because of its potent apoptotic activity, PUMA has been considered as a promising therapeutic molecule for cancer treatment (Ito et al., 2005). In addition, the therapeutic potential of targeted PUMA inhibition to suppress apoptosis for the treatment of degenerative diseases has attracted considerable research interest (Yu and Zhang, 2008). However, our current findings suggest that Puma gene delivery or inhibition may lead to undesired effects on vascular and immune systems and warrant more careful investigations.
Although we have demonstrated an unexpected function of PUMA using comprehensive systems and approaches, outstanding questions remain. For example, we currently cannot explain how PUMA regulates cytosolic calcium levels and Erk activation. Also, it remains elusive how the functional property of PUMA is regulated to fulfill its autophagy or apoptotic activities in different cells or under different conditions. It is possible that there may be intermediate context-dependent molecules mediating the different activities of PUMA. Moreover, although our data demonstrated a cell-autonomous function of PUMA in microglia and vascular cells, this does not rule out the possibility that programed elimination of certain types of retinal cells (e.g., neurons) by PUMA during development might signal the proliferation of retinal microglia and vascular cells. Future studies are needed to answer these questions. In summary, our data revealed an angiogenic activity of PUMA, which can act as a survival and proliferative factor by modulating autophagy, and demonstrated an unexpected complexity of PUMA function.
Experimental Procedures
Animals, Intraocular Injections, and Tissues
The mice used and the eye tissue isolation are described in the Extended Experimental Procedures. For Puma knockdown in vivo, 2 μg/0.5 μl/eye of pLKO.1 DNA plasmid encoding the mouse Puma shRNA or the pLKO.1 empty vector (Open Biosystems) together with the in vivo-jetPEI reagent (Polyplus-transfection) was injected into P1 mouse vitreous according to the manufacturer's instructions. For Puma gene delivery in vivo, 2 μg/0.5 μl/eye of pCMV6-AC-RFP vector encoding mouse PUMA or the pCMV6-AC-RFP vector without PUMA (OriGene Technologies) with the in vivo transfection reagent was injected into P1 mouse vitreous. In vivo microglia depletion was described previously (Checchin et al., 2006).
Immunofluorescent Staining and Retinal Image Analysis
Immunofluorescent staining and retinal image analysis are described in the Extended Experimental Procedures. The reagents used for staining were: isolectin GS-B4 (IB4, Invitrogen), DAPI (Invitrogen), rabbit anti-mouse CD11b (BD PharMingen), rat anti-CD31 (PharMingen), rabbit anti-iba1 (Wako), rabbit anti-PUMA (Cell Signaling), goat anti-mouse PDGFRα (R&D), mouse anti-GFAP (Sigma-Aldrich), rabbit anti-LC3B (Novus Biologicals), and the Alexa Fluor fluorescence-conjugated IgGs (Invitrogen).
Western Blot and Real-Time PCR
Western blot was performed as described (Tang et al., 2010). The antibodies used were: rabbit anti-PUMA (Cell Signaling, Sigma-Aldrich), rabbit anti-FGF2 (Santa Cruz), rat anti-IL6 (Biolegend), armenian hamster anti-CCL2 (Biolegend), rabbit anti-LC3B (Novus Biologicals), rabbit anti-M-CSFR (Cell Signaling), mouse anti-p44/42 MAPK (Erk1/2) (Cell Signaling), and rabbit anti-Phospho-p44/42 MAPK (Erk1/2) (Cell Signaling). Monoclonal anti-β-actin-peroxidase (Sigma-Aldrich) was used as an internal control. Real-time PCRs were performed in triplicates as described (Tang et al., 2010) and repeated at least once. Primers used are listed in Table S3. Primers Used for Real-Time PCR, Related to Figures 1, 2, and 3.
Cornea Pocket and Aortic Ring Assays
The mouse cornea pocket assay was performed as described (Li et al., 2008; Zhang et al., 2009). Human VEGF165 (PeproTech) of 160 ng/pellet/cornea was used in the experiments. CD11b+ microglia density within the active angiogenic area was assessed using an Imager Z1 microscope and AxioVision software (Carl Zeiss). The aortic ring assay was performed as described (Kumar et al., 2010) using human VEGF165 and FGF2 (100 ng/ml, R&D Systems). Images of the aortic rings were taken on day 7 after implantation and microvessels analyzed as described (Kumar et al., 2010). CD11b+ cells were analyzed by immunostaining.
Laser-Induced Choroidal Neovascularization Model
The CNV model was performed as described (Zhang et al., 2009). Eight to ten-week-old mice were used for experiments. A total of 1 μg/eye of pLKO.1 mouse Puma shRNA plasmid or the control pLKO.1 plasmid without the Puma shRNA (Open Biosystems) together with the transfection reagent in vivo-jetPEI (Polyplus-transfection) was injected into the mouse vitreous according to the manufacturer's instructions. CNV area was analyzed 1 week after laser treatment using IB4 staining. Microglia density was analyzed by immunofluorescence staining using Iba1 as a marker.
Primary Cell Isolation and Cell Culture
Primary mouse retina and brain microglia cells, astrocyte, brain ECs, and aortic artery SMCs were isolated and cultured as described (Matsubara et al., 2000; Saijo et al., 2009; Seidel et al., 1991). Cells within ten passages were used for experiment. The immortalized TR-iBRB and RGC5 were cultured as described (Li et al., 2008). The HUVEC (Lonza) and RAW264.7 macrophage cells (ATCC) were cultured according to the manufacturer's instructions.
Intracellular Calcium Assay
The intracellular calcium assay was performed according to the manufacturer's instructions (Invitrogen) and is described in the Extended Experimental Procedures. The primary retinal microglia cells transfected with a pRFP-C-RS vector encoding the mouse Puma shRNA or the pRFP-C-RS vector encoding a noneffective 29-mer scrambled shRNA (OriGene Technologies) were used for experiments 3 days after transfection.
Proliferation, Survival, and Cell Chemotaxis Assay
Several methods were used to assess cell proliferation and survival, including an MTT assay, a DAPI-TUNEL staining method, a BrdU assay, a trypan blue exclusion assay, and a LIVE/DEAD viability/cytotoxicity assay (Invitrogen). Primary cells isolated from six to eight mice were pooled and cultured for experiments. Serum-free medium was used for EC, SMC, and 293FT cell starvation. The Hank's balanced salt solution (Invitrogen) was used for microglia starvation. For the MTT assay, experiments were performed according to the manufacturer's protocol (Invitrogen). The Erk inhibitor A6355 and Mek inhibitor U0126 (10 μM each, Sigma) were used to block the Erk pathway using 0.1% DMSO (Sigma-Aldrich) as a control. A DAPI (Invitrogen) and a TUNEL (Roche) staining method was used to count the number of viable and apoptotic cells in 24 fields (450 μm × 350 μm each) each assay and the mean value calculated. Experiments were performed in triplicate under a normoxic or hypoxic condition (1% oxygen) and repeated at least once, and representative data presented. For the BrdU proliferation assay, cells were cultured in complete medium and cell proliferation assessed using a cell proliferation ELISA BrdU colorimetric kit according to the manufacturer's protocol (Roche). For cell survival assay, primary retinal microglia cells were treated with mitomycin C (Sigma, 50 μg/ml) in serum-free medium for 2 hr to inhibit cell proliferation and then cultured in serum-free medium with repeated mitomycin C treatment every 24 hr. After 3 days, the viable cells were counted using a trypan blue exclusion method. The viable and dead cells were also stained and analyzed using a LIVE/DEAD viability/cytotoxicity assay kit (Invitrogen). The pLKO.1 Puma shRNA plasmid and the control pLKO.1 plasmid without the Puma shRNA (Open Biosystems), and the pCMV6 Puma gene expression plasmid and the control pCMV6 plasmid without the Puma gene (OriGene Technologies), were transfected into the cells using a SuperFect reagent (QIAGEN) according to the manufacturer's protocol. Puromycin (Sigma) or G418 (Sigma) was used for selection according to the manufacturer's protocol. Three biological repeats were performed for each experiment and the mean value from the repeats is presented. Microglia chemotaxis assay was performed as described (Ma et al., 2009) using the chemoattractants ATP (50 μM, Sigma), CCL2 (100 ng/ml, R&D), and CCL3 (50 ng/ml, R&D).
Statistical Analysis
Two-tailed Student's t test was used for statistical analysis. Difference was considered statistically significant when p < 0.05. Results are represented as mean ± SEM of the number of determinations.
Animals, Intraocular Injections, and Tissues
All animal experiments were approved by the Animal Care and Use Committee at the NEI/NIH (NEI-553), and were performed according to the NIH regulations. Puma- and Noxa-deficient mice were described previously (Kieran et al., 2007; Villunger et al., 2003). Mice were bred on C57Bl6 background for more than six generations and littermates were used for the experiments. Cx3cr1GFP/GFP mice (The Jackson Laboratory) were used for breeding to obtain the Cx3cr1GFP/+Puma−/− and Cx3cr1GFP/+Puma+/+ littermates. Intravitreal injections were performed using 33G needles as described previously (Li et al., 2008).
Immunofluorescent Staining and Retinal Image Analysis
The mice were euthanized and the eyes harvested and fixed in 4% paraformaldehyde. The retinae and corneas were dissected from the eyes for whole-mount staining, or the eyes and brains were OCT-embedded and cryosectioned at 12 μm thickness. For immunofluorescence staining, the samples were washed in PBS containing 0.1% Triton X-100, blocked with 5% BSA (Sigma-Aldrich) with 5% goat serum (Invitrogen), and then stained with different antibodies or markers. Image analysis was performed using a Carl Zeiss Imager Z1 or a LSM510 confocal microscope. The image-processing softwares used were AxioVision (Carl Zeiss), Photoshop CS (Adobe), and Image J 1.44 (NIH). The vascular area, blood vessel branch point, vessel diameter, microglia, and astrocyte density were measured as described previously (Connor et al., 2007; Hellström et al., 2007). Forty to fifty microscopic fields each sample were analyzed. The retinal thickness was measured as described (Komeima et al., 2007).
Intracellular Calcium Assay
For primary retinal microglia cells, right after the initial imaging to assess steady-state intracellular calcium level, an ATP (1mM, Sigma) stimulation was applied and subsequent time-lapse Ca2+ levels assessed by imaging the cells every 6.5 s for about 6 min. For the vascular endothelial and smooth muscle cells, right after the initial imaging to assess the steady-state intracellular calcium level, a mechanical stimulation was applied by pipetting the culture medium for 6∼7 s, and the subsequent time-lapse intracellular calcium level assessed by imaging the cells every 1 s for about 30 s. Fluorescence intensity in the cells of interest at the peak time point during time-lapse imaging was quantified, and the ratio of maximum fluorescence intensity change after stimulation over the initial baseline fluorescence was calculated and used as the Fluo-4AM Index.
Function of the PUMA-RFP Construct
The function of the PUMA-RFP construct was tested using HEK293FT cells, which have been commonly used in the field to study the function of PUMA (Sandow et al., 2012). PUMA-RFP overexpression induced apoptosis in the HEK293FT cells (Figures S5A and S5B, n = 3, p < 0.01), consistent with the previous observation by other investigators using a different PUMA expression vector (Sandow et al., 2012). Thus, the PUMA-RFP construct retains a traditional apoptotic function of PUMA.
In Vivo Microglia Depletion
A total of 0.5 μl of clodronate liposomes was prepared using phosphatidylcholine (Sigma-Aldrich), and cholesterol (Sigma-Aldrich) was injected into the mouse vitreous within 24 hr of birth to deplete the resident retinal microglia. An additional 4 μl of clodronate liposomes was injected into the peritoneal to deplete systemic microglia/macrophages.
Measurement of Retinal Thickness
For the measurement of the thickness of different retinal layers, the whole eyes were sectioned. The sections were stained using the hematoxylin and eosin method. Three stained sections, on which the optic nerve was visible, were photographed. The thickness of the different retinal layers were measured using three sections at six locations of each section: 25% (S1), 50% (S2), and 75% (S3) of the distance between the superior pole and the optic nerve, and 25% (I1), 50% (I2), and 75% (I3) of the distance between the inferior pole and the optic nerve using a Carl Zeiss microscope and an AxioVision software (Carl Zeiss). Each location was measured three times on three sections, and the mean value presented. For the total retinal thickness, the mean value of all the measurements (3 × 3 × 6 = 54) was presented.
Primary Microglia Cell Isolation and Culture
The retinae or cerebrum of the brains were dissected from six to eight P1 neonatal mice. The meninges were removed from the cerebrum. Tissues were digested with 0.4% trypsin (Invitrogen) for 30 min at 37°C and gently dissociated using a BD Falcon Cell Strainer (BD Biosciences). The resulting cell suspension was centrifuged and resuspended in DMEM-F12 complete medium (DMEM/F12, 5% heat-inactivated fetal bovine serum, 5% horse serum and 1% penicillin/streptomycin from Invitrogen). The cells were seeded in poly-L-lysine (Sigma-Aldrich) coated tissue culture flasks and incubated at 37°C. The medium was changed every 3 days. After 14 to 21 days of incubation, the microglia cells were isolated from the cultured cells by shaking the flasks on an orbital shaker for 1 hr at 300 rpm at 4°C and then cultured in the complete medium at 37°C with 5% CO2.
Primary Vascular Endothelial Cell Isolation and Culture
The brain cerebral tissues were dissected from six to eight P1 neonatal mice and rinsed in ice-cold PBS three times. The tissues were minced and digested with 10 ml PBS containing collagenase A (1 mg/ml, Sigma), DNase I (0.05 mg/ml, Sigma), 0.05% trypsin-EDTA (Invitrogen) for 30 min at 37°C. The solution was filtered using a BD Falcon cell strainer (BD Biosciences) and centrifuged at 1,000 g for 5 min. The cell pellet was re-suspended in 1 ml DMEM medium with 10% fetal bovine serum (FBS) and 20% mouse serum to block the Fc receptors for 30 min at room temperature. The cells were washed twice with ice-cold DMEM medium containing 10% FBS and resuspended in 1 ml ice-cold DMEM medium containing 10% FBS and incubated with the sheep anti-rat IgG-conjugated Dynabeads (Invitrogen) that were preincubated with rat anti-mouse CD31 antibody (BD PharMingen). The endothelial cells were then isolated according to the manufacturer's instruction and seeded on flasks precoated with 1% gelatin (Sigma-Aldrich) and cultured in endothelial cell medium (EGM-2 BulletKit, Lonza) at 37°C with 5% CO2.
Western Blot and Real-Time PCR
For western blot, protein extracts from tissues or cells were prepared using the RIPA buffer (Sigma-Aldrich). Western blot was performed using an XCell western blot system according to the manufacturer's instructions (Invitrogen). Samples were electrophoresed under a reducing condition on a 10% or 4%–12% SDS-PAGE gradient gel (Invitrogen). Proteins on the gel were transferred to a polyvinylidene difluoride membrane (Invitrogen). The membranes were blocked with 5% BSA (Sigma-Aldrich) and incubated with different antibodies. For real-time PCR, total RNA was isolated using a RNeasy Mini-kit (QIAGEN) according to the manufacturer's instruction. A total of 3 μg of total RNA was used for cDNA synthesis using the SuperScript SuperMix kit (Invitrogen). Real-time PCR was performed using an ABI Prism 7500 HT Sequence Detection System (Applied Biosystems).
Intracellular Calcium Assay
For the intracellular calcium assay, primary retinal microglia, primary vascular endothelial and smooth muscle cells were seeded in a glass bottom dish (MatTek Corporation) and incubated in Hank's buffered salt solution (HBSS) containing 4 μM of the Ca2+ indicator Fluo-4AM (Invitrogen), 10 μM of pluronic F-127 (Invitrogen), and 0.1% OxyFluor (Oxyrase) for 30 min at room temperature. After 30 min, the cells were washed and incubated with Ca2+-free HBSS. Fluorescence images were taken to analyze the steady-state calcium level and time-lapse calcium changes using a LSM 510 multiphoton microscopy.
We thank Dr. Andreas Strasser at the Walter and Eliza Hall Institute of Medical Research, Australia, for the Puma- and Noxa-deficient mice, and Professor Birgitta Öhberg at the Linköping University, Sweden, for the great help with the zebrafish work. T.C. was supported by the ERC (ENDHOMRET) and the Deutsche Forschungsgemeinschaft. H.F.L. was supported by the Volkswagen foundation (Lichtenberg program) and the Deutsche Forschungsgemeinschaft (KFO 274). Y.C. is supported by the Swedish Research Council, the Swedish Cancer Foundation, the Karolinska Institute Foundation, the Karolinska Institute Distinguished Professor Award, the Tianjin Natural Science Foundation (CMM-Tianjin, 09ZCZDSF04400), the Torsten Soderbergs Foundation, the European Union Integrated Project of Metoxia (222741), and the European Research Council (ERC) ANGIOFAT (250021). The authors declare no conflict of interest. This research was supported by the Intramural Research Program of the NIH, National Eye Institute.
Supplemental Information
- Table S1. Puma Is Highly Expressed in Myeloid and Vascular Cells, as well as in Other Human Tissues and Cells, Related to Figure 1 (XLSX 12 kb)
- Myeloid and vascular cells are in bold. Approximate expression patterns inferred from EST sources of online bioinformatics database UniGene (http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Mm.7660).
- Table S2. Puma Is Highly Expressed in Macrophages and Microglia, as well as in Other Mouse Tissues and Cells, Related to Figure 1 (XLSX 12 kb)
- Macrophages and microglia are in bold. Approximate expression patterns inferred from EST sources of online bioinformatics database UniGene (http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Mm.7660).
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Publication Information
Received: December 16, 2011
Revised: June 5, 2012
Accepted: September 24, 2012
Published: November 1, 2012


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Undercover executives get an up-close and personal look at both the good and the bad. Occasionally, they discover incredible unsung heroes, people who make their companies great. And at times, the mission uncovers mediocre performances and disappointing attitudes.
The show's fifth season begins Friday, and I'm still not sure why I like it so much. Perhaps at some level I enjoy watching under-par performers get their just desserts. You have to admit, there is a bit of sweet justice when a jerk comes face to face with the boss who saw them for who they really are. On the other hand, it is inspiring to see an unsung hero discovered and rewarded for loyalty and sacrificial service.
As followers of Christ, we have the ultimate undercover boss! Jesus spoke often about the "Father who sees what is done in secret" (Matthew 6:6) and who examines the motives of the heart. Psalm 139 speaks of the One who knows when I sit down and when I stand up, and knows my every thought from afar. The Father even knows what I am going to say before I say it. Then the psalmist concludes that such knowledge is too great for him to comprehend.
The Father is ever-present and all-knowing. Perhaps that's why the apostle Paul urged the believers in the church at Colossae, "Whatever you do, work at it with all your heart, as working for the Lord, not for men" (Colossians 3:23), and to the Corinthians he said, "So whether you eat or drink or whatever you do, do it all for the glory of God" (1 Corinthians 10:31).
On the show, people put their jobs and future on the line by not recognizing that their boss is right in front of them and is watching and listening to how they treat customers and co-workers. In the same way, the ultimate undercover boss is always listening, always watching and always aware of the unspoken attitudes and feelings in our hearts.
Paul reminded his readers, "So we make it our goal to please Him, whether we are at home, in the body or away from it. For we must all appear before the judgment seat of Christ, that each one may receive what is due him for the things done while in the body, whether good or bad" (2 Corinthians 5:9-10).
In Matthew 25, Jesus talks about of the separation of the sheep and goats at the final judgment (my paraphrase): I tell you the truth, whatever you did (or did not do) for one of the least of these my brothers, you did (or did not do) for Me.
What would you do or say differently if you knew the person with whom you are speaking today is, in fact, the Creator and Sustainer of the Universe -- the ultimate undercover boss?
Larry Doyle is director of missions of the Piedmont Baptist Association in North Carolina. Get Baptist Press headlines and breaking news on Twitter (
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Keith Lamarr Hamilton
Position: DT-DE
Height: 6-6 Weight: 295 lbs.
Born: May 25, 1971 in Paterson, NJ
College: Pittsburgh (school history)
Drafted by the New York Giants in the 4th round (99th overall) of the 1992 NFL Draft.
Weighted Career AV (100-95-...): 72 (496th overall since 1950)
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Output includes all games from 1940 to 2013.


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BeerAdvocate on your phone?! True story. Try the beta now.
Tickets for American Craft Beer Fest are on sale now! (May 30 & 31 in Boston, MA)
Berserker Imperial Stout - Midnight Sun Brewing Co.
Displayed for educational use only; do not reuse. |
Ratings: 651 | Reviews: 216 | Show All Ratings:
4.29/5 rDev -1.2%
look: 4.5 | smell: 4.25 | taste: 4.25 | feel: 4.5 | overall: 4.25
Poured from a 22oz bottle
A: Oily black with a really good looking dark brown head
S: Bourbon, bitter dark chocolate
T: Really rich flavors of bourbon and bitter dark chocolate dominate, hints of espresso and molasses more at the end. Well hidden 12.7% alcohol
M: So thick and so good
O: I think this is better than a lot of the most highly rated bourbon barrel stouts out there. Lots of really well balanced and strong flavors
Serving type: bottle
08-04-2013 01:21:28 | More by anteater
3.43/5 rDev -21%
look: 4.5 | smell: 4.25 | taste: 3 | feel: 4 | overall: 3
Not sure what year this is, no sign of a date. I believe 2012? 12.7% ABV
A: Thick pitch black pour. Big dense dark brown head. Very dense. Head away in a very dense and fluffy manner (if that makes sense). Heavy walls of lacing. Great retention.
S: Interesting. Mellow bourbon with burnt sugar and caramel. Oaky. Heavy, sweet molasses. Dark chocolate. When swirled I get a strong fruity wine aroma. Maybe a bit of savory umami in there too? A lot going on.
T: Very interesting flavor. It's bourbon and wine battling it out in the beginning. Dark fruits and almost tart berries duking it out with burnt sugars, toffee and molasses. Some chocolate and char mixing in too. Chalky charred oak in the finish with hints of smoke. Light chocolate and umami...burnt meat really... lingers between sips. Red wine really layers over everything once it's warm. Pretty boozy.
M: Big, heavy, thick, etc. A bit chalky.
O: I rarely say this, but almost a little too much going on here. Roasted malt of this stout takes a backseat to lots of bourbon barrel character, wine barrel character and molasses. I prefer a strong roasted malt character in my stouts, myself. Did not get any maple syrup in this one either oddly enough. Having said that, very complex and surprisingly balanced. Just not quite my forte, especially since I'm not a huge red wine fan and this one has a surprising amount of it.
Serving type: bottle
07-07-2013 01:39:16 | More by Dope
3.85/5 rDev -11.3%
look: 4 | smell: 4 | taste: 4 | feel: 3.5 | overall: 3.5
650 mL bottle poured into a snifter. Reviewed from notes. 2012 bottle.
Appearance - Almost inky. Good sized mocha head. Collar of dark khaki foam as it settles.
Smell - Bourbon is nice but faint. Slight vinous characteristic. Oak and some dark fruits combining, probably, but it's a little more acidic than that. Lots of chocolate.
Taste - Nice chocolate. Bitter dark chocolate. Molasses. Bourbon is even fainter here. Bitter finish, seemingly from roasted malts.
Mouthfeel - Extremely bitter, not what I expected. Masks a lot of flavor. Well carbonated.
Overall - The barrel seems weak to me, but the other flavors are good.
Serving type: bottle
06-10-2013 17:05:15 | More by Alieniloquium
4.56/5 rDev +5.1%
look: 4 | smell: 4.5 | taste: 4.75 | feel: 4.25 | overall: 4.5
Picked this up in a great trade with Mike! Pitch black pour with a beautiful caramel-colored one finger head with decent retention and nice lacing. Big roasty nose with hints of bourbon, molasses, vanilla, oak, cocoa, and some interesting vinous notes and some subtle maple. The taste is just as bold and amazing with hints of dark chocolate, port wine, vanilla, bourbon, mocha, molasses, and some subtle black cherry and maple. The mouthfeel is thick, creamy, and mouth-coating with medium carbonation and a nice bitter dry finish. Overall, this was one surprising beer. Very complex, but not overdone. Will definitely seek it out again!
Serving type: bottle
05-24-2013 19:45:49 | More by beerdrifter
4.91/5 rDev +13.1%
look: 4.75 | smell: 4.75 | taste: 5 | feel: 4.75 | overall: 5
A black as night, khaki head, nice lacing, beautiful!
S vanilla, chocolate, bourbon, maple, light hop, caramel, white chocolate, sweet vermouth,
T vanilla, white chocolate, sweet vermouth, salted caramel, big bourbon and whiskey flavors, omg Is it good!
My hick, viscous, delicious!
O holy shot. This is amazing! One of the best liquids ever.
Serving type: bottle
05-17-2013 02:23:31 | More by Lare453
4.44/5 rDev +2.3%
look: 4 | smell: 4.5 | taste: 4.75 | feel: 4 | overall: 4
A - Dark black with a finger of frothy dark chocolate colored head. Fades fairly slowly.
S - Roasted chocolate malts, maple syrup, molasses, Bourbon, oak, vanilla, dark fruits, black licorice, tobacco, and booze.
T - Dark chocolate malts, maple syrup, molasses, coffee, oak, vanilla, Bourbon, dark fruits, black licorice, tobacco, and some booze.
M - Full bodied with moderate carbonation. Smooth and a bit thick with a warming and sweet finish.
D - Great Bourbon barrel aged imperial stout. The maple syrup is a great touch.
Serving type: bottle
04-30-2013 05:01:34 | More by ngeunit1
New York
3.93/5 rDev -9.4%
look: 4.5 | smell: 4.5 | taste: 3.5 | feel: 4.25 | overall: 4
Served in a snifter glass.
Appearance: A thick opaque brown, medium bubbles on the body...pours like straight motor oil thick and the head...dark tan, rising tojust about a finger's length.
Smell: Heavy brown sugar maybe the molasses, very sticky...all you need to do is smell it once and it's with you forever
Taste: A straight blend of molasses and brown sugar, it is a little too sweet for my part. I for one am for bitterness with my stouts period. This brew lacks it and just reading the label I noticed it's just at 30 IBU's...nothing wrong with that but seriously where is the rest of the taste.
Mouth: Sticky sugar with medium carbonation. What it lacks in taste makes up for it in stickiness but depends how much if it you want. Best served with dessert.
Overall: This brew improves within time of the consumption of the glass, the proiblem is the sweetness might overwhelm some including myself. If you like imperial stouts which I absolutely love this is definitely something a little different and not for everyone. Try a glass just to try it...Salud!
Serving type: bottle
04-18-2013 02:44:59 | More by BlurryVisi0n
4.01/5 rDev -7.6%
look: 4.75 | smell: 4 | taste: 4 | feel: 3.75 | overall: 4
22 oz bottle, 2012 vintage, from trade with beergod1 - thanks man. Poured into a Half Acre snifter at cellar temp.
A: Almost motor-oil black with a very dark tan head of an impressive 2 fingers with great retention and lacing.
S: Maple is the predominant smell, a little bourbon, a little sweetness, perhaps from molasses, not much roasty character or chocolate.
T: A little bland at first, not a lot of maple or molasses. Barrel characters of oak and vanilla come in late, and a bourbon finish on the back of the tongue. Not as much malt character as I'd like from a stout of this magnitude. Aside from the bourbon taste, not too much heat. Pretty drinkable.
M: A little over-carbonated, and not as thick as I'd like. Doesn't really coat the tongue at all.
O: Definitely disappointing, maybe its just the 2012 vintage that everyone has been complaining about, but this doesn't really impress at all, tastes like a standard stout with some bourbon. Might give 2013 a try next year but won't have the 2012 again.
Serving type: bottle
03-19-2013 01:03:13 | More by MCain04
New York
4.35/5 rDev +0.2%
look: 4 | smell: 4.25 | taste: 4.5 | feel: 4.25 | overall: 4.25
A: The beer is absolutely jet black in color. It poured with a thin ring of reddish mocha bubbles around the edge of the glass that quickly died down, leaving only a faint collar.
S: Strong aromas of dark malts and bourbon are present in the nose, along with some hints of molasses.
T: The taste is similar to the smell, but a little more complex with additional flavors of dark malts and chocolate.
M: It feels medium- to full-bodied and viscous on the palate with a low to moderate amount of carbonation. A little warming from the alcohol is noticeable in the finish.
O: The flavors of this beer are well integrated and balanced, particularly the bourbon, which does not overpower the beer and overshadow the other flavors.
Serving type: bottle
03-17-2013 01:29:08 | More by metter98
North Carolina
4.31/5 rDev -0.7%
look: 4 | smell: 4.75 | taste: 4.25 | feel: 4 | overall: 4.25
2012 Edition
A: Pours an opaque and thick jet black in color with very light notes of visible carbonation and some dark reddish brown highlights. The beer has an eighth of a finger tall dark reddish mocha head which quickly reduces to a thin ring at the edges of the glass. Light amounts of lacing are observed.
S: The beer has a moderate to strong aroma of chocolate and bourbon with some lighter hints of oak and vanilla. There are moderate notes of molasses and maple syrup sweetness. Light notes of roasted malts.
T: Upfront there are some light notes of roasted malts with some molasses and maple syrup sweetness. On top of the malts, there are some moderate to strong flavors of rich dark chocolate and bourbon with some lighter notes oak and vanilla. Light hints of cocoa with some hints of alcohol towards the finish. Light lingering bitterness.
M: Full bodied with moderate amounts of carbonation. Moderate amounts of alcohol warming in the finish.
O: This is a great beer to finish the evening and matches very well with dessert - in our case some brownies made with Sea Legs Baltic Porter. This is really rich and you can detect the alcohol but I'm going to enjoy sipping on it a while longer. The bourbon is well integrated into the beer and the flavors in the beer match really well.
Serving type: bottle
03-17-2013 01:21:57 | More by dbrauneis
British Columbia (Canada)
4.18/5 rDev -3.7%
look: 4.25 | smell: 4 | taste: 4.25 | feel: 4 | overall: 4.25
2012 Bottle at Brouwer's cafe.
Appearance. Pours thick and black with a skim of tan head.
Smell. Molasses, wood, black licorice, rum and dark malts with a little fudge.
Taste. Chocolate fudge and molasses, toasted wood and licorice root, dark and maybe lightly smokey malts.
Mouthfeel. Full body, maybe on the lower side. Boozy warmth and low+ carbonation.
Overall. Nice big stout. Didn't get much maple, but very tasty and lots of molasses.
Serving type: bottle
02-24-2013 04:18:25 | More by Rutager
New York
4.76/5 rDev +9.7%
look: 4.5 | smell: 5 | taste: 4.75 | feel: 4.5 | overall: 4.75
Knocking out this long-time want courtesy of huskermike12, who sent it along with a box of awesomeness in the Super Bowl XLVII Squares BIF...
A: Fairly aggresive pour into a CBS snifter produces a thick, 2-finger light brown head. Head retention is excellent, especially compared to most barrel-aged stouts that pour with little to no head to begin with. Body is deep black, which makes the pour look like a slice of chocolate cake with whipped chocolate mousse on top.
S: First sniff is HUGE chocolate with a wisp of bourbon on the tail end. Reminds me of a warm chocolate lava cake. Cant wait to dive into this.
T: With chocolate still assaulting my nose, the first sip is dominated by cocoa, followed by the slightest cameo appearance of maple and finishing with a restrained dose of bourbon. Taste definitely delivers on what the nose promised. The barrel adds the perfect amount of bourbon, complementing the chocolate without ever dominating the taste. This is like a bourbon spiked hot cocoa. Touches of coffee, molasses ad fudge throughout. The one drawback, the maple never made a reappearance after the first sip.
M: Thick as a brick. Coats the tongue, tastebuds and everything in between. Not sure if this is part of what the molasses adds, but if so, it should be a requirement for every imperial stout.
O: Loved it! The one disappointment was the absence of the maple, but I have yet to find a beer that fully delivers on the maple promise (going to have to find me some Lawsons next). Other than that, I would stockpile this if it was readily available. Going to have to steal some of my son's Nestle Quick, toss it in a coffee mug, microwave it and spike it with some bourbon to hold me over til the next time I can get my hands on some of this.
Serving type: bottle
02-24-2013 03:17:55 | More by ant880
4.49/5 rDev +3.5%
look: 4.25 | smell: 4.5 | taste: 4.5 | feel: 4.5 | overall: 4.5
22oz bottle poured into large snifter - thanks to brystmar - 2012 bottle
Viscous opaque blackness in the glass with a bubbled dark brownie colored head resting on top. Thick collar of tanned retention with spotty lacing left on the glass.
Smoky and sweet in the nose, driven by a combination of maple syrup, chocolate cake and bourbon. Oak, dark fruit, coffee and vanilla bean with toffee and roasted dark malts. Warm and boozy.
Slick and sweet up front with bourbon warmth, cocoa powder and dark chocolate bark. Vanilla beans with caramel and toffee, layered on a maple syrup base. Oaky and warm with a thick, robust malt backbone. Touches of tobacco and red wine with roasted coffee beans. Multiple layers of flavor and malt continue to evolve while drinking, leading into a boozy and sweet finish.
Full bodied, medium carbonation, creamy and sticky on the tongue. I've tasted this a couple times before, but this was my first time enjoying the better part of a bottle and I couldn't be happier with this. Wonderfully deep and warm with plenty of rich bourbon and chocolate, finished off with sticky maple syrup and vanilla. I'd seek this out again in a second.
Serving type: bottle
02-23-2013 04:16:45 | More by xnicknj
4.29/5 rDev -1.2%
look: 4 | smell: 4.5 | taste: 4.25 | feel: 4.25 | overall: 4.25
2012 bomber into a 3F wine-style glass. Big thanks to Tony.
A: Pitch black body with a thin head of fine brown foam that settles into a ring and leaves some delicate lace. Nice.
S: Sweet, roasty, oaky, spiritous, and rich. Dark chocolate, bourbon, char, raisins, and molasses. Dark and intense.
T: Follows the nose. Crazy collage of intense flavors: molasses, red wine, grape skin, bourbon, tobacco, dark chocolate, charcoal, and boozy bitterness. A little much, but really enjoyable.
M: Rich, full body with lots of carbonation. Maybe a little too frothy, but pleasant.
O: Really enjoyable, but a little rough. I'm loving a third of the bottle, but I can't imaging drinking a whole one.
Serving type: bottle
02-01-2013 02:54:16 | More by woosterbill
4.24/5 rDev -2.3%
look: 4 | smell: 4.25 | taste: 4.25 | feel: 4.25 | overall: 4.25
Much thanks to Ben for this one!
A: Pours a thick black with a dark tan head on top, decent retention forming a nice cap on tip. Moderate lacing.
S: This beer has a very nice aroma, some bourbon and roasted malt with a bit of chocolate and deep molasses. There is a little bit of smoke on the aroma as well.
T: Lots going on in the flavor profile here. Some chocolate and a bit of coffee upfront with a definite maple sweetness mixed in. There is a little bit of dark fruit, mostly raisin and cherry. Also a touch of smoke.
M: Full bodied, definitely on the thick side. Rather chewy.
O: A very solid beer from Midnight Sun. A beer I was happy to have split, not sure how much of it I could drink on my own.
Serving type: bottle
01-30-2013 16:37:22 | More by mdaschaf
4.38/5 rDev +0.9%
look: 4.5 | smell: 5 | taste: 4 | feel: 4.5 | overall: 4.5
Pours a viscus black, dark tan head, frothy, lacy, and creamy.
Aroma of coffee, fudge, vanilla, oak, bourbon, booze, and chocolate covered raisins.
Roasted taste, with cocoa, and touches of espresso. Light bourbon, with maple-y finish, oak and sap. Alcohol is there, but svelt all things considered.
It's vicious. Thick, chewy, and syrupy. Coats, sticks and has epic substance.
Can't deny this pedigree!
Serving type: bottle
01-01-2013 06:35:55 | More by EgadBananas
4.43/5 rDev +2.1%
look: 4.5 | smell: 4.5 | taste: 4.5 | feel: 4.25 | overall: 4.25
A-black as night, completely opaque. Half-finger dark brown head that remains.
S-lots of toffee, maple, wood, bourbon,
T-dark chocolate and oak up front. more subtle bourbon tones follow with a dry finish.
M-little to no carbonation and very rich. Thick beer
O-Really nice offering here. Although they may be different styles, I prefer arctic devil to this one. The alcohol does catch up on this one and is pretty hard to have more than a small pour.
Serving type: bottle
12-16-2012 18:51:34 | More by beerhan
4.58/5 rDev +5.5%
look: 3.5 | smell: 4.5 | taste: 5 | feel: 3.5 | overall: 4.5
Bomber with huge thanks to Boozecamel for sharing. Reviewed from notes.
Motor-oil consistency and hue. Modest dark tan head, average retention, recedes to a film.
Pungent and cutting earthy maple syrup, chocolate brownies, oak, beef jerky, molasses. Nice dryness and subtle nuttiness.
Simultaneously sweet and dry, which is an impressive coup. Most of theh aroma elements are there to some degree, although dominant flavours are thick choc-carmael fudge and liquorice. Amazing.
Thick and viscous, however carbonation strikes me as aggressive for the style.
Full-on and uncompromising with a great flavour/aroma profile. Enough said.
Serving type: bottle
12-14-2012 01:14:26 | More by spicelab
3.85/5 rDev -11.3%
look: 4 | smell: 4 | taste: 4 | feel: 3.5 | overall: 3.5
Thanks Blake!
Not sure on the vintage, I believe this is the latest so 2012? Maybe 2011.
Pour was black, little head. Pretty thick looking
Dark chocolate and roasted charred malt. Some sweet bitterness.
Lots of char and roast. Light sweetness, but well blended with the bitterness. Hints of dark chocolate and coffee.
A bit lighter than it looks, mild carbonation.
Pretty decent, nothing really mind blowing.
Serving type: bottle
12-10-2012 15:03:20 | More by hooliganlife
3.35/5 rDev -22.8%
look: 4 | smell: 4 | taste: 3 | feel: 3 | overall: 3.5
2012 vintage
appearance: served in an oversized snifter, jet black body, creamy mocha lace
smell: bourbon, tannin notes, rich fudge
taste: charred malt, fudge, smoke, light bourbon, soy sauce. I don't know if its the "infection" or what, but at $20, this is a huge dissapointment.
mouthfeel: a bit thin, lots of soy in the finish
overall: this is a Pass in the future
Serving type: bottle
12-09-2012 22:09:55 | More by dirtylou
4.58/5 rDev +5.5%
look: 4 | smell: 5 | taste: 4.5 | feel: 4.5 | overall: 4.5
On rumors of a possible infection I cracked my first of four bottles tonight before it potentially starts going downhill.
22oz bottle (2012) into a tulip. Pours a light-sucking obsidian black with a gorgeous reddish-brown head, about 1 finger, that settles to a thin film and ring.
Aroma is rich and enticing. Heavy bourbon and oak, roasty dark malt, earthy nuts, sticky molasses. An elusive hint of the wine barrels that grows stronger as it warms.
Taste follows nose. Deep and complex. Bourbon, roasted malt, molasses, dark fruit, oak, chocolate, smoky char. Nice balance between strong bitterness and high-gravity sweetness. Alcohol is completely hidden.
Mouthfeel is heavy, sticky, slick. Just about right for a beer of this magnitude.
Overall, just an overpowering bourbon-soaked beast of a beer. Hopefully the infection rumors aren't true but if they are I'll enjoy drinking the remainder before they're too far gone.
Serving type: bottle
12-06-2012 06:17:24 | More by draheim
4.58/5 rDev +5.5%
look: 4 | smell: 4.5 | taste: 4.5 | feel: 4.5 | overall: 5
22oz bottle pours a pitch/tar black with a thin light-brown head. Some moderate carbonation and light lacing as it moves down the glass.
Smell is smoke, booze, burnt sugar/molasses, and maple. Hint of winter fruit.
Taste is dark fruit (raison/plum), maple syrup, charred wood, burnt sugar. Some tobacco flavor emerges as it rolls around the mouth. Cherry in the finish. Some booze flavor from the bourbon oak barrel aging. Very complex.
Mouthfeel is thick, chewy , and viscous. Velvety smooth with just a hint of carbonation. Not too sweet or syrupy. Just right.
Overall, a massive, complex, contemplative beer. Agree with comments below about letting it warm some. Really opens up further and provides additional nuances to explore. Take a deep breath, relax, and enjoy.
Serving type: bottle
12-06-2012 05:28:41 | More by JohnnyP3
4.28/5 rDev -1.4%
look: 4 | smell: 4 | taste: 4.5 | feel: 4.5 | overall: 4
Many, many thanks to paulys55 for this bottle!
A - Poured pitch black with about 1/4 of creamy, toffee colored head and no carbonation.
S - Charred/smoked oak tannins, tobacco peat, dark fruit, molasses, dark roasted chocolate, and mild alcohol presence (for such a big beer).
T - Smoke, oak, maple, bourbon, molasses, roasted bitter chocolate, dark cherries, and alcohol effervescence.
M - Thick, viscous, sticky, slightly syrupy, sticky.
O - Letting this beer sit in the glass for 30+ minutes and warming to room temperature really opened this up in an incredibly complex way. In fact, if I ever have the opportunity to enjoy another bottle of this I will most likely pop it at room temperature instead, far better.
Serving type: bottle
12-03-2012 00:28:45 | More by jzeilinger
New York
4.55/5 rDev +4.8%
look: 4.5 | smell: 4.5 | taste: 4.5 | feel: 5 | overall: 4.5
22oz Bottle split two-way
Solid brown color, deep and unfeathered. Tan head sticks with the glass and moves nicely, not cloyed.
Rich aroma, chocolate, malt, slightly booze. This was poured in a couple of ~4 oz rounds for each of us and the second pour had considerably more booze in the nose. Plum or candied plum.
Exceptional flavor. Deep with raisin base. Chocolate notes with apropriate bitterness, oak and an alcohol bite. There is also a touch or anise or another spice I can't put my finger on that rounds everything together. Missing the dark fruit/plum that is a large component of the aroma.
Viscosity is perfect. Solid body which is well carbonated. It is noteworthy that the flavor ends with a crispness often absent from RIS. Perfect or damn near it.
There is a lot going on here and it is fair to say this is in the top handful of personal favorites. Easily able to drink it over the course of a sitting and would happily enjoy it as often as I could locate another bottle. For the style it meshes an uncommon lightness in consumption with a full mouthfeel and requisite abv.
Serving type: bottle
12-01-2012 21:43:18 | More by CooperEllis
4.18/5 rDev -3.7%
look: 4.5 | smell: 4.5 | taste: 4 | feel: 4.5 | overall: 4
22 oz. bottle served in a Midnight Sun Chub. 2012 vintage. Huge thanks to BA mfavoino for sending this my way. Thanks Matt!
A: Jet black body with a thick and creamy dark tan head forming on the pour. It recedes slowly to a thick cap and collar. Sticky sheets of lace are left on the glass.
S: Big molasses sweetness with smooth bourbon nose. Dark chocolate, oak and some nice roasted malts compliment the sweetness. The smell is delicious. Extremely impressed so far.
T: Unfortunately the taste isn't quite as nice up front. Very sweet maple syrup with some bourbon and a big roasted malt presence. The booze is a little harsh and the oak barrel isn't as well integrated as the nose would suggest. The finish is creamy chocolate and sweet molasses. Some brown sugar and vanilla notes come out as it warms.
M: Heavy bodied with a nice chew to it. Oily and viscous, definitely a sipper here. Medium low carbonation.
O: The nose was great and at the moment the barrel is a little too disjointed from the base beer. Some time will do this beer well to subside the boozy notes and harsh oak.
Serving type: bottle
11-27-2012 01:31:36 | More by Treebs
Berserker Imperial Stout from Midnight Sun Brewing Co.
out of 100
based on 651 ratings.


text:
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The Minnesota Water Resources Conference presents innovative and practical water resource engineering solutions, management techniques, and current research about Minnesota's water resources. The conference provides an opportunity to address: 1) lessons learned from the implementation of engineering projects; 2) best practices discovered in the design and application of water resource management techniques, 3) implications of water policy decisions, and 4) research into current and emerging issues. The conference facilitates interaction among engineers, water resources managers, researchers, and local, state and federal agency staff.
Minnesota Water Resources Conference For more information, visit www.wrc.umn.edu/waterconf
Event Id: 186519, , 10/16/2012 - 10/17/2012, RiverCentre, Ballroom - 175 Kellogg Blvd, St Paul, M


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With towering pillars and sweeping arches, the lobby at Paragon Chateau 14 resembles an official monument to the pleasures of moviegoing. Sony 4K HD digital projection systems flash current-run films onto each screen. In addition to a fully-stocked concession stand, the theater hosts The Lot, a lounge where moviegoers can order beer, wine, and soda served in hollowed-out Golden Globes and listen to live music.
The sails of the 31-foot sloop Messenger wave in the wind during Sail Pepin's outings. The staff and crew man the vessel for fun family outings, romantic cruises, sailing lessons, and team-building programs. At their location in the historic Breakwater Building on Lake Pepin's waterfront, they also entertain guests with a wine bar, coffee shop, and live music.
Anytime Fitness, which boasts 1,689 clubs in North America, makes it easier for average folks to etch out time for exercise by doing one simple thing: staying open 24 hours a day for 365 days a year. As fitness seekers challenge themselves on cardio and Hammer Strength machines and hoist Iron Grip free weights in clean, well-stocked facilities, security monitoring ensures they're safe and producing enough sweat to meet official government standards. Members can also ramp up their exercise regimens with the help of Anytime Fitness's staff of personal trainers, who demonstrate moves and sling motivating tips. After workouts, guests can shower in the private restrooms or hop into one of the tanning booths available 24 hours a day.
Formerly farmland for five generations of the Schmidt family, Lake Pepin Golf Course now boasts 18 holes that span across rugged links-style topography flanked by the bluffs of the Mississippi River Valley. The course plays to 6,752 yards, with 12 holes showcasing panoramic views of the lake and the remaining six holes providing access to an underground miniature golf course. Those who prefer manmade scenery can gawk at Joe "Snowball" Schmidt's 1869 two-story stone house, nestled between the 17th green and 18th tee.
Several years ago, Ken Smith and Chase Williams were typical door-to-door salesmen. In need of a gimmick to boost sales and a quicker mode of transport, the duo tried riding Segways and immediately fell in love. Eventually, they turned their love for the two-wheeled transporter into their current venture, All American Segway Tours. Their company provides all manner of Segway engagement, including guided Segway tours that offer rolling educations in local history and wildlife. Customers can also discover the revolutionary devices on their own by renting out Segways for recreational use, private parties, or as a friend for a lonely office chair or shopping cart.


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House fire sparks on Garland Street in Clifton
Posted: 5:14 PM Jan 17, 2013
Reporter: KKCO 11 News
House fire on Garland Street in Clifton
KKCO 11 News has crews on scene at a house fire on the 500 block of Garland Street in Clifton.
There are two Grand Junction Fire Department trucks and two Clifton Fire Department trucks on scene.
The family that lives in the home tells KKCO 11 News that no one was in the house at the time of the fire, but they were home just ten minutes before the incident.
No injuries have been reported.
We do not yet know the extent of the damage.
Stick with KKCO 11 News for the latest information on this developing story as we get it.
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Brett Comer scored 21 points and Florida Gulf Coast claimed the first NCAA tournament berth in school history, knocking off top-seeded Mercer 88-75 in the Atlantic Sun championship game Saturday.
The second-seeded Eagles (24-10) became the first team in the 68-team field by snapping Mercer's 17-game home winning streak, tied for the sixth-longest in the nation. Sherwood Brown added 16 points and 11 rebounds as Florida Gulf Coast pulled away after a back-and-forth first half.
Brown sparked a 14-3 spurt that broke open the tight game, sending Florida Gulf Coast to the NCAAs in just their sixth season as a Division I program. The Eagles celebrated at midcourt, hugging and jumping around after getting their championship gear.
FGCU will now sit back and wait as the rest of the NCAA field is set over the next 8 days, culminating in the NCAA Selection Show next Sunday.


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