Role of Endomucin in Hypoxia-Induced Retinopathy of Prematurity

Retinopathy of prematurity (ROP) is a major cause of blindness among premature, low birth weight infants as a result of pathological angiogenesis. Angiogenesis, the growth of new blood vessels from preexisting vessels, occurs in the veins and capillaries of the body. The process is highly regulated during early development and maturation. However, under abnormal conditions such as a decrease in oxygen levels or hypoxia, angiogenesis can become dysregulated and pathogenic. Currently, the best treatment for ROP is laser therapy, which does not significantly improve vision. Alternatively, glycoproteins are believed to play an important role in angiogenesis. Endomucin (EMCN), a glycoprotein, has been shown to be expressed by the venous and capillary endothelium. EMCN is believed to be associated with angiogenesis and could be a potential target for treatment of ROP. Thus, we hypothesize that EMCN is regulated by hypoxia and plays an important role in pathological angiogenesis. Human retinal endothelial cells (HRECs), representative of endothelial cells involved in retinal angiogenesis, were deprived of oxygen using a hypoxia chamber. We established the optimal oxygen dosage, determined the optimal cell density, and monitored EMCN expression at different time points after exposure to hypoxia. Changes in gene expression in response to hypoxia were compared to control cells. Our preliminary data indicates that EMCN is regulated by hypoxia. Currently, we are investigating whether EMCN has similar effects in regulating revascularization in vivo. Taken together, our study indicates a novel role for EMCN during hypoxia-induced angiogenesis which may serve as a therapeutic target

AAV-CRISPR/Cas9–Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro

Purpose Pathologic angiogenesis is a component of many diseases, including neovascular age-related macular degeneration, proliferation diabetic retinopathy, as well as tumor growth and metastasis. The purpose of this project was to examine whether the system of adeno-associated viral (AAV)–mediated CRISPR (clustered regularly interspaced short palindromic repeats)–associated endonuclease (Cas)9 can be used to deplete expression of VEGF receptor 2 (VEGFR2) in human vascular endothelial cells in vitro and thus suppress its downstream signaling events. Methods: The dual AAV system of CRISPR/Cas9 from Streptococcus pyogenes (AAV-SpGuide and -SpCas9) was adapted to edit genomic VEGFR2 in primary human retinal microvascular endothelial cells (HRECs). In this system, the endothelial-specific promoter for intercellular adhesion molecule 2 (ICAM2) was cloned into the dual AAV vectors of SpGuide and SpCas9 for driving expression of green fluorescence protein (GFP) and SpCas9, respectively. These two AAV vectors were applied to production of recombinant AAV serotype 5 (rAAV5), which were used to infect HRECs for depletion of VEGFR2. Protein expression was determined by Western blot; and cell proliferation, migration, as well as tube formation were examined. Results: AAV5 effectively infected vascular endothelial cells (ECs) and retinal pigment epithelial (RPE) cells; the ICAM2 promoter drove expression of GFP and SpCas9 in HRECs, but not in RPE cells. The results showed that the rAAV5-CRISPR/Cas9 depleted VEGFR2 by 80% and completely blocked VEGF-induced activation of Akt, and proliferation, migration as well as tube formation of HRECs. Conclusions: AAV-CRISRP/Cas9–mediated depletion of VEGFR2 is a potential therapeutic strategy for pathologic angiogenesis

Gq/11-Mediated Signaling and Hypertrophy in Mice with Cardiac-Specific Transgenic Expression of Regulator of G-Protein Signaling 2

Cardiac hypertrophy is a well-established risk factor for cardiovascular morbidity and mortality. Activation of $G_{q/11}$-mediated signaling is required for pressure overload-induced cardiomyocyte (CM) hypertrophy to develop. We previously showed that among Regulators of G protein Signaling, RGS2 selectively inhibits $G_{q/11}$ signaling and its hypertrophic effects in isolated CM. In this study, we generated transgenic mice with CM-specific, conditional RGS2 expression (dTG) to investigate whether RGS2 overexpression can be used to attenuate $G_{q/11}$-mediated signaling and hypertrophy in vivo. Transverse aortic constriction (TAC) induced a comparable rise in ventricular mass and ANF expression and corresponding hemodynamic changes in dTG compared to wild types (WT), regardless of the TAC duration (1-8 wks) and timing of RGS2 expression (from birth or adulthood). Inhibition of endothelin-1-induced $G_{q/11}$-mediated phospholipase C β activity in ventricles and atrial appendages indicated functionality of transgenic RGS2. However, the inhibitory effect of transgenic RGS2 on $G_{q/11}$-mediated PLCβ activation differed between ventricles and atria: (i) in sham-operated dTG mice the magnitude of the inhibitory effect was less pronounced in ventricles than in atria, and (ii) after TAC, negative regulation of $G_{q/11}$ signaling was absent in ventricles but fully preserved in atria. Neither difference could be explained by differences in expression levels, including marked RGS2 downregulation after TAC in left ventricle and atrium. Counter-regulatory changes in other $G_{q/11}$-regulating RGS proteins (RGS4, RGS5, RGS6) and random insertion were also excluded as potential causes. Taken together, despite ample evidence for a role of RGS2 in negatively regulating $G_{q/11}$ signaling and hypertrophy in CM, CM-specific RGS2 overexpression in transgenic mice in vivo did not lead to attenuate ventricular $G_{q/11}$-mediated signaling and hypertrophy in response to pressure overload. Furthermore, our study suggests chamber-specific differences in the regulation of RGS2 functionality and potential future utility of the new transgenic model in mitigating $G_{q/11}$ signaling in the atria in vivo

Endomucin inhibits VEGF-induced endothelial cell migration, growth, and morphogenesis by modulating VEGFR2 signaling

Angiogenesis is central to both normal and pathologic processes. Endothelial cells (ECs) express O-glycoproteins that are believed to play important roles in vascular development and stability. Endomucin-1 (EMCN) is a type I O-glycosylated, sialic-rich glycoprotein, specifically expressed by venous and capillary endothelium. Evidence has pointed to a potential role for EMCN in angiogenesis but it had not been directly investigated. In this study, we examined the role of EMCN in angiogenesis by modulating EMCN levels both in vivo and in vitro. Reduction of EMCN in vivo led to the impairment of angiogenesis during normal retinal development in vivo. To determine the cellular basis of this inhibition, gain- and loss-of-function studies were performed in human retinal EC (HREC) in vitro by EMCN over-expression using adenovirus or EMCN gene knockdown by siRNA. We show that EMCN knockdown reduced migration, inhibited cell growth without compromising cell survival, and suppressed tube morphogenesis of ECs, whereas over-expression of EMCN led to increased migration, proliferation and tube formation. Furthermore, knockdown of EMCN suppressed VEGF-induced signaling as measured by decreased phospho-VEGFR2, phospho-ERK1/2 and phospho-p38-MAPK levels. These results suggest a novel role for EMCN as a potent regulator of angiogenesis and point to its potential as a new therapeutic target for angiogenesis-related diseases

Transgenic RGS2 Protein is expressed in a cardiac-specific, tTA-dependent, Dox-regulatable manner and is functionally active.

<p>[<b>A</b>] Representative Western blot of crude ventricular homogenates (10 µg/lane) from 3-months old double transgenes (dTG) of two independent transgenic RGS2 lines (L1, L12). The blot was probed with antibodies recognizing RGS2 and GAPDH, which was used as loading control. [<b>B</b>–<b>D</b>] Western blots of crude ventricular homogenates from indicated genotypes obtained from Line 1 matings (<u>B</u>), of crude organ homogenates from dTG mice only (<u>C</u>), and membrane and cytosolic fractions from dTG ventricles (<u>D</u>). Each blot was probed with an anti-FLAG antibody. Calsequestrin expression and Ponceau S stain were used as controls (panels B and C/D, respectively). The arrow in panel D denotes a lane loaded with a molecular weight marker. [<b>E</b>] Total inositol phosphate (IP) formation in ventricles from RGS2 TG (open bars, n = 3) and dTG mice (n = 4 each; closed bars). Freshly dissected tissue pieces were incubated for 30 min in the absence (basal) or presence of endothelin-1 (ET-1, 100 nM). # P<0.05 dTG vs RGS2 TG. [<b>F</b>] H&E stained four-chamber sections of hearts from a 12-week-old wild-type (WT) and dTG (L1) mouse. [<b>G</b>] Quantitative Western blot analysis of crude ventricular homogenates from dTG mice: (a) RGS2 protein suppression within 4 days after Doxycycline (Dox, 100 mg/kg) administration. (b) RGS2 re-expression in mice, which had received Dox (100 mg/kg) from gestation until 3 weeks of age, after indicated weeks of withdrawal from Dox (closed bars, n = 4 each); untreated 11 week-old dTG controls (- Dox, open bar, n = 5) are shown for comparison.</p

TAC and Ang II induce chamber-specific down-regulation of transgenic RGS2 protein and mRNA.

<p>[<b>A</b>] Representative Western blot of crude ventricular homogenates (60 µg/lane) from WT and dTG (L1) ventricles after sham or 8 wk TAC, which were probed with antibodies recognizing RGS2 (1 and 10 sec exposure times) and GAPDH. [<b>B</b>] Ventricular RGS2 protein downregulation in dTG mice (L1, conditional transgene expression) after TAC for 1 wk (n = 9) or 8 wks (n = 15) compared to respective sham controls (n = 7–9). * <i>P</i><0.05 TAC vs Sham. [<b>C</b>] Ventricular RGS2 mRNA downregulation in dTG mice (L1, conditional transgene expression) after TAC for 1 wk (n = 6) or 8 wks (n = 12) compared to respective sham controls (n = 6–8). The effect of Ang II infusion (n = 4) compared to vehicle controls (n = 2) for 12 days is also shown. * <i>P</i><0.05 TAC vs Sham or Ang II vs vehicle respectively. [<b>D</b>] Ventricular RGS2, RGS4 and RGS5 mRNA expression in WT and dTG (L1) mice 8 wks after TAC or sham operation. Real-time PCR data normalized to 18S are expressed relative to respective WT sham (n = 7–10 in each group, except for n = 3 for RGS2 in WT). [<b>E</b>] <i>Left:</i> Representative Western blot of crude homogenates (30 µg/lane) of L12 and L1 dTG ventricles (V) and atrial appendages (A) probed with antibodies recognizing RGS2 and GAPDH. <i>Right:</i> Inhibition of ET-1-induced IP formation in left atrial appendages (LA) and ventricles (LV) from sham-operated L12 (n = 6–7) and L1 (n = 5) dTG. Data are expressed in % of the ET-1-induced response in WT (set at 100%, not shown). # <i>P</i><0.05 dTG vs WT (set at 100%). [<b>F</b>] RGS2 mRNA expression in left and right ventricles and atrial appendages from dTG L12 mice (constitutive transgene expression) 10 days after TAC compared to respective sham controls (n = 5–7). * <i>P</i><0.05 TAC vs Sham.</p

Transgenic RGS2 expression <i>in vivo</i> differentially inhibits G_q/11-mediated PLCβ activation in ventricles and atrial appendages.

<p>Basal (Bas) and Endothelin-1 (ET-1)-induced (100 nM, 30 min) total inositol phosphate (IP) formation in left and right ventricles [<b>A</b>] as well as left and right atrial appendages [<b>B</b>] from WT mice (<i>open bars</i>) and dTG mice (L1, constitutive RGS2 expression, <i>closed bars</i>) 6 wks after sham-operation (<i>cross-hatched bars,</i> n = 5 WT and dTG each) or TAC (<i>solid bars</i>, n = 4 WT and n = 6 dTG). Data are expressed in % of basal values in WT for each region (panel A: 18–25 cpm/mg tissue for LV and RV after sham or TAC; panel B: 100-140 and 55–85 cpm/mg for LA and RA after sham or TAC, respectively). *<i>P</i><0.05 ET-1 vs basal; #<i>P</i><0.05 dTG vs WT (ET-1 effect); ̂P<0.05 dTG vs WT (basal). The right panels (b) depict the inhibition of ET-1-induced IP formation (i.e., difference in IP formation in the presence or absence of ET-1 in the respective panel a) in indicated regions of dTG mice. Data are expressed in % of the ET-1-induced response in WT (set at 100%, not shown). #<i>P</i><0.05 dTG vs WT.</p

dTG and WT mice have comparable hypertrophic and functional responses to TAC and Ang II.

<p>[<b>A</b>] <u>Hypertrophic response to pressure overload (<i>panel a</i>) and Ang II infusion (<i>panel b</i>):</u> Ventricular weight (normalized to body weight, <i>top panel</i>) and ANF mRNA (normalized to 18S and expressed relative to sham controls, <i>bottom panel</i>) in wild-type mice (WT) and double transgenic mice (dTG, L1) with conditional or constitutive RGS2 transgene expression after transverse aortic constriction (TAC, n = 10–18 WT and n = 11–15 dTG) or Ang II infusion (n = 9 each) for indicated durations (<i>solid bars</i>). Animals subjected to sham operation (n = 9–15 WT and n = 7–9 dTG) or vehicle (saline, n = 5–6 each) were used for comparison (<i>cross-hatched bars</i>). ANF expression was assessed at least in half of the animals in each group. * <i>P</i><0.05 TAC vs sham (a) or Ang II vs vehicle (b); # <i>P</i><0.05 dTG vs WT. [<b>B</b>] <i><u>In vivo</u></i><u> hemodynamic assessment of left ventricular function:</u> (a) Representative pressure volume (PV) recordings during preload reduction via inferior vena cava compression in WT mice that had been subjected to TAC or sham operation 4 wks earlier. The traces show raw data before subtraction of volume from parallel conduction. The solid lines indicate end-systolic pressure volume relationship (ESPVR). (b) Group data obtained from PV loop analyses for indicated parameters in WT and dTG mice with conditional RGS2 expression (L1) after TAC for 1 wk (n = 11 WT and n = 8 dTG) or 8 wks (n = 13–15 WT and n = 8–9 dTG) compared to respective sham controls (1 wk: n = 9 WT and n = 5 dTG; 8 wks: n = 7–8 WT and n = 5–6 dTG). * <i>P</i><0.05 TAC vs Sham; # <i>P</i><0.05 dTG vs wt.</p