Serine-arginine-rich protein kinase-1 inhibition for the treatment of diabetic retinopathy.

Angiogenic VEGF isoforms are upregulated in diabetic retinopathy (DR), driving pathological growth and fluid leakage. Serine-arginine-rich protein kinase-1 (SRPK1) regulates VEGF splicing, and its inhibition blocks angiogenesis. We tested the hypothesis that SRPK1 is activated in diabetes, and an SRPK1 inhibitor (SPHINX31) switches VEGF splicing in DR and prevents increased vascular permeability into the retina. SRPK1 was activated by high glucose (HG), in a PKC-dependent manner, and was blocked by SPHINX31. HG induced release of SRSF1 from the nuclear speckles, which was also SRPK1 dependent, and increased retinal pigment epithelial (RPE) monolayer admittance, which was reversed by SRPK1 inhibition (P < 0.05). Diabetes increased retinal permeability and thickness after 14 days which was blocked by treatment with SPHINX31 eye drops (P < 0.0001). These results show that SRPK1 inhibition, administered as an eye drop, protected the retinal barrier from hyperglycemia-associated loss of integrity in RPE cells in vitro and in diabetic rats in vivo. A clinical trial of another SRPK1 inhibitor has now been initiated in patients with diabetic macular edema.NEW & NOTEWORTHY VEGF-A165b splicing is induced by hyperglycemia through PKC-mediated activation of SRPK1 in RPE cells, increasing their permeability and angiogenic capability. SRPK1 inhibitors can be given as eye drops to reduce retinal permeability and edema in diabetic retinopathy.

Measurement of Revascularization in the Hind Limb After Experimental Ischemia in Mice.

Peripheral vascular disease is a major cause of morbidity and mortality, and is a consequence of impaired blood flow to the limbs. This arises due to the inability of the tissue to develop sufficiently functional collateral vessel circulation to overcome occluded arteries, or microvascular impairment. The mouse hind limb model of hind limb ischemia can be used to investigate the impact of different treatment modalities, behavioral changes, or genetic knockout. Here we described the model in detail, providing examples of adverse events, and details of ex vivo analysis of blood vessel density.

Enhanced notch signaling modulates unproductive revascularization in response to nitric oxide-angiopoietin signaling in a mouse model of peripheral ischemia.

INTRODUCTION: Arteriolargenesis can be induced by concomitant stimulation of nitric Oxide (NO)-Angiopoietin receptor (Tie)-Vascular Endothelial Growth Factor (VEGF) signaling in the rat mesentery angiogenesis assay. We hypothesized that the same combination of exogenously added growth factors would also have a positive impact on arteriolargenesis and, consequently, the recovery of blood flow in a model of unilateral hindlimb ischemia. RESULTS AND METHODS: NO-Tie mice had faster blood flow recovery compared to control mice, as assessed by laser speckle imaging. There was no change in capillary density within the ischemic muscles, but arteriole density was higher in NO-Tie mice. Given the previously documented beneficial effect of VEGF signaling, we tested whether NO-Tie-VEGF mice would show further improvement. Surprisingly, these mice recovered no differently from control, arteriole density was similar and capillary density was lower. Dll4 is a driver of arterial specification, so we hypothesized that Notch1 expression would be involved in arteriolargenesis. There was a significant upregulation of Notch1 transcripts in NO-Tie-VEGF compared with NO-Tie mice. Using soluble Dll4 (sDll4), we stimulated Notch signaling in the ischemic muscles of mice. NO-Tie-sDll4 mice had significantly increased capillary and arteriole densities, but impaired blood flow recovery. CONCLUSION: These results suggest that Dll4 activation early on in revascularization can lead to unproductive angiogenesis and arteriolargenesis, despite increased vascular densities. These results suggest spatial and temporal balance of growth factors needs to be perfected for ideal functional and anatomical revascularisation.

The adipokine leptin modulates adventitial pericyte functions by autocrine and paracrine signalling.

Transplantation of adventitial pericytes (APCs) improves recovery from tissue ischemia in preclinical animal models by still unknown mechanisms. This study investigates the role of the adipokine leptin (LEP) in the regulation of human APC biological functions. Transcriptomic analysis of APCs showed components of the LEP signalling pathway are modulated by hypoxia. Kinetic studies indicate cultured APCs release high amounts of immunoreactive LEP following exposure to hypoxia, continuing upon return to normoxia. Secreted LEP activates an autocrine/paracrine loop through binding to the LEP receptor (LEPR) and induction of STAT3 phosphorylation. Titration studies using recombinant LEP and siRNA knockdown of LEP or LEPR demonstrate the adipokine exerts important regulatory roles in APC growth, survival, migration and promotion of endothelial network formation. Heterogeneity in LEP expression and secretion may influence the reparative proficiency of APC therapy. Accordingly, the levels of LEP secretion predict the microvascular outcome of APCs transplantation in a mouse limb ischemia model. Moreover, we found that the expression of the Lepr gene is upregulated on resident vascular cells from murine ischemic muscles, thus providing a permissive milieu to transplanted LEP-expressing APCs. Results highlight a new mechanism responsible for APC adaptation to hypoxia and instrumental to vascular repair.

Type-2 diabetes increases autophagy in the human heart through promotion of Beclin-1 mediated pathway.

BACKGROUND: Diabetes promotes progressive loss of cardiac cells, which are replaced by a fibrotic matrix, resulting in the loss of cardiac function. In the current study we sought to identify if excessive autophagy plays a major role in inducing this progressive loss. METHODS AND RESULTS: Immunofluorescence and western blotting analysis of the right atrial appendages collected from diabetic and non-diabetic patients undergoing coronary artery bypass graft surgery showed a marked increase in the level of autophagy in the diabetic heart, as evidenced by increased expression of autophagy marker LC3B-II and its mediator Beclin-1 and decreased expression of p62, which incorporates into autophagosomes to be efficiently degraded. Moreover, a marked activation of pro-apoptotic caspase-3 was observed. Electron microscopy showed increased autophagosomes in the diabetic heart. In vivo measurement of autophagic flux by choloroquine injection resulted in further enhancement of LC3B-II in the diabetic myocardium, confirming increased autophagic activity in the type-2 diabetic heart. Importantly, in-vitro genetic depletion of beclin-1 in high glucose treated adult rat cardiomyocytes markedly inhibited the level of autophagy and subsequent apoptotic cell death. CONCLUSIONS: These findings demonstrate the pathological role of autophagy in the type-2 diabetic heart, opening up a potentially novel therapeutic avenue for the treatment of diabetic heart disease.

Data supporting the activation of autophagy genes in the diabetic heart.

This data article contains full list of autophagy related genes that are altered in diabetic heart. This article also shows data from in vitro cultured cardiomyocytes that are exposed the high glucose treatment to simulate hyperglycemic state in vitro. The interpretation of these data and further extensive insights into the regulation of SG biogenesis by AMPK can be found in "Type-2 diabetes increases autophagy in the human heart through promotion of Beclin-1 mediated pathway" (Munasinghe et al., in press) [1].

p75(NTR)-dependent activation of NF-κB regulates microRNA-503 transcription and pericyte-endothelial crosstalk in diabetes after limb ischaemia.

The communication between vascular endothelial cells (ECs) and pericytes in the microvasculature is fundamental for vascular growth and homeostasis; however, these processes are disrupted by diabetes. Here we show that modulation of p75(NTR) expression in ECs exposed to high glucose activates transcription of miR-503, which negatively affects pericyte function. p75(NTR) activates NF-κB to bind the miR-503 promoter and upregulate miR-503 expression in ECs. NF-κB further induces activation of Rho kinase and shedding of endothelial microparticles carrying miR-503, which transfer miR-503 from ECs to vascular pericytes. The integrin-mediated uptake of miR-503 in the recipient pericytes reduces expression of EFNB2 and VEGFA, resulting in impaired migration and proliferation. We confirm operation of the above mechanisms in mouse models of diabetes, in which EC-derived miR-503 reduces pericyte coverage of capillaries, increased permeability and impaired post-ischaemic angiogenesis in limb muscles. Collectively, our data demonstrate that miR-503 regulates pericyte-endothelial crosstalk in microvascular diabetic complications.

Expansion and characterization of neonatal cardiac pericytes provides a novel cellular option for tissue engineering in congenital heart disease.

BACKGROUND: Living grafts produced by combining autologous heart-resident stem/progenitor cells and tissue engineering could provide a new therapeutic option for definitive correction of congenital heart disease. The aim of the study was to investigate the antigenic profile, expansion/differentiation capacity, paracrine activity, and pro-angiogenic potential of cardiac pericytes and to assess their engrafting capacity in clinically certified prosthetic grafts. METHODS AND RESULTS: CD34(pos) cells, negative for the endothelial markers CD31 and CD146, were identified by immunohistochemistry in cardiac leftovers from infants and children undergoing palliative repair of congenital cardiac defects. Following isolation by immunomagnetic bead-sorting and culture on plastic in EGM-2 medium supplemented with growth factors and serum, CD34(pos)/CD31(neg) cells gave rise to a clonogenic, highly proliferative (>20 million at P5), spindle-shape cell population. The following populations were shown to expresses pericyte/mesenchymal and stemness markers. After exposure to differentiation media, the expanded cardiac pericytes acquired markers of vascular smooth muscle cells, but failed to differentiate into endothelial cells or cardiomyocytes. However, in Matrigel, cardiac pericytes form networks and enhance the network capacity of endothelial cells. Moreover, they produce collagen-1 and release chemo-attractants that stimulate the migration of c-Kit(pos) cardiac stem cells. Cardiac pericytes were then seeded onto clinically approved xenograft scaffolds and cultured in a bioreactor. After 3 weeks, fluorescent microscopy showed that cardiac pericytes had penetrated into and colonized the graft. CONCLUSIONS: These findings open new avenues for cellular functionalization of prosthetic grafts to be applied in reconstructive surgery of congenital heart disease.

Combined intramyocardial delivery of human pericytes and cardiac stem cells additively improves the healing of mouse infarcted hearts through stimulation of vascular and muscular repair.

RATIONALE: Optimization of cell therapy for cardiac repair may require the association of different cell populations with complementary activities. OBJECTIVE: Compare the reparative potential of saphenous vein-derived pericytes (SVPs) with that of cardiac stem cells (CSCs) in a model of myocardial infarction, and investigate whether combined cell transplantation provides further improvements. METHODS AND RESULTS: SVPs and CSCs were isolated from vein leftovers of coronary artery bypass graft surgery and discarded atrial specimens of transplanted hearts, respectively. Single or dual cell therapy (300 000 cells of each type per heart) was tested in infarcted SCID (severe combined immunodeficiency)-Beige mice. SVPs and CSCs alone improved cardiac contractility as assessed by echocardiography at 14 days post myocardial infarction. The effect was maintained, although attenuated at 42 days. At histological level, SVPs and CSCs similarly inhibited infarct size and interstitial fibrosis, SVPs were superior in inducing angiogenesis and CSCs in promoting cardiomyocyte proliferation and recruitment of endogenous stem cells. The combination of cells additively reduced the infarct size and promoted vascular proliferation and arteriogenesis, but did not surpass single therapies with regard to contractility indexes. SVPs and CSCs secrete similar amounts of hepatocyte growth factor, vascular endothelial growth factor, fibroblast growth factor, stem cell factor, and stromal cell-derived factor-1, whereas SVPs release higher quantities of angiopoietins and microRNA-132. Coculture of the 2 cell populations results in competitive as well as enhancing paracrine activities. In particular, the release of stromal cell-derived factor-1 was synergistically augmented along with downregulation of stromal cell-derived factor-1-degrading enzyme dipeptidyl peptidase 4. CONCLUSIONS: Combinatory therapy with SVPs and CSCs may complementarily help the repair of infarcted hearts.

Epigenetic profile of human adventitial progenitor cells correlates with therapeutic outcomes in a mouse model of limb ischemia.

OBJECTIVE: We investigated the association between the functional, epigenetic, and expressional profile of human adventitial progenitor cells (APCs) and therapeutic activity in a model of limb ischemia. APPROACH AND RESULTS: Antigenic and functional features were analyzed throughout passaging in 15 saphenous vein (SV)-derived APC lines, of which 10 from SV leftovers of coronary artery bypass graft surgery and 5 from varicose SV removal. Moreover, 5 SV-APC lines were transplanted (8×10(5) cells, IM) in mice with limb ischemia. Blood flow and capillary and arteriole density were correlated with functional characteristics and DNA methylation/expressional markers of transplanted cells. We report successful expansion of tested lines, which reached the therapeutic target of 30 to 50 million cells in ≈10 weeks. Typical antigenic profile, viability, and migratory and proangiogenic activities were conserved through passaging, with low levels of replicative senescence. In vivo, SV-APC transplantation improved blood flow recovery and revascularization of ischemic limbs. Whole genome screening showed an association between DNA methylation at the promoter or gene body level and microvascular density and to a lesser extent with blood flow recovery. Expressional studies highlighted the implication of an angiogenic network centered on the vascular endothelial growth factor receptor as a predictor of microvascular outcomes. FLT-1 gene silencing in SV-APCs remarkably reduced their ability to form tubes in vitro and support tube formation by human umbilical vein endothelial cells, thus confirming the importance of this signaling in SV-APC angiogenic function. CONCLUSIONS: DNA methylation landscape illustrates different therapeutic activities of human APCs. Epigenetic screening may help identify determinants of therapeutic vasculogenesis in ischemic disease.

Rapid onset of cardiomyopathy in STZ-induced female diabetic mice involves the downregulation of pro-survival Pim-1.

BACKGROUND: Diabetic women are five times more likely to develop congestive heart failure compared with two fold for men. The underlying mechanism for this gender difference is not known. Here we investigate the molecular mechanisms responsible for this female disadvantage and attempt safeguarding cardiomyocytes viability and function through restoration of pro-survival Pim-1. METHODS AND RESULTS: Diabetes was induced by injection of streptozotocin in CD1 mice of both genders. Functional and dimensional parameters measurement using echocardiography revealed diastolic dysfunction in female diabetic mice within 8 weeks after STZ-induced diabetes. This was associated with significant downregulation of pro-survival Pim-1 and upregulation of pro-apoptotic Caspase-3, microRNA-1 and microRNA-208a. Male diabetic mice did not show any significant changes at this time point (P < 0.05 vs. female diabetic). Further, the onset of ventricular remodelling was quicker in female diabetic mice showing marked left ventricular dilation, reduced ejection fraction and poor contractility (P < 0.05 vs. male diabetic at 12 and 16 weeks of STZ-induced diabetes). Molecular analysis of samples from human diabetic hearts confirmed the results of pre-clinical studies, showing marked downregulation of Pim-1 in the female diabetic heart (P < 0.05 vs. male diabetic). Finally, in vitro restoration of Pim-1 reversed the female disadvantage in diabetic cardiomyocytes. CONCLUSIONS: We provide novel insights into the molecular mechanisms behind the rapid onset of cardiomyopathy in female diabetics. These results suggest the requirement for the development of gender-specific treatments for diabetic cardiomyopathy.

Perivascular delivery of encapsulated mesenchymal stem cells improves postischemic angiogenesis via paracrine activation of VEGF-A.

OBJECTIVE: To test the therapeutic activity of perivascular transplantation of encapsulated human mesenchymal stem cells (MSCs) in an immunocompetent mouse model of limb ischemia. APPROACH AND RESULTS: CD1 mice underwent unilateral limb ischemia, followed by randomized treatment with vehicle, alginate microbeads (MBs), MB-encapsulated MSCs (MB-MSCs), or MB-MSCs engineered with glucagon-like peptide-1. Treatments were applied directly in the perivascular space around the femoral artery. Laser Doppler and fluorescent microsphere assessment of blood flow showed a marked improvement of perfusion in the MB-MSCs and MB-MSCs engineered with glucagon-like peptide-1 groups, which was associated with increased foot salvage particularly in MB-MSCs engineered with glucagon-like peptide-1-treated mice. Histological analysis revealed increased capillary and arteriole density in limb muscles of the 2 MSC groups. Furthermore, MB-MSCs engineered with glucagon-like peptide-1 and, to a lesser extent, MB-MSC treatment increased functional arterial collaterals alongside the femoral artery occlusion. Analysis of expressional changes in ischemic muscles showed that MB-MSC transplantation activates a proangiogenic signaling pathway centered on vascular endothelial growth factor A. In contrast, intramuscular MB-MSCs caused inflammatory reaction, but no improvement of reparative vascularization. Importantly, nonencapsulated MSCs were ineffective either by intramuscular or perivascular route. CONCLUSIONS: Perivascular delivery of encapsulated MSCs helps postischemic reperfusion. This novel biological bypass method might be useful in patients not amenable to conventional revascularization approaches.

Transplantation of human pericyte progenitor cells improves the repair of infarcted heart through activation of an angiogenic program involving micro-RNA-132.

RATIONALE: Pericytes are key regulators of vascular maturation, but their value for cardiac repair remains unknown. OBJECTIVE: We investigated the therapeutic activity and mechanistic targets of saphenous vein-derived pericyte progenitor cells (SVPs) in a mouse myocardial infarction (MI) model. METHODS AND RESULTS: SVPs have a low immunogenic profile and are resistant to hypoxia/starvation (H/S). Transplantation of SVPs into the peri-infarct zone of immunodeficient CD1/Foxn-1(nu/nu) or immunocompetent CD1 mice attenuated left ventricular dilatation and improved ejection fraction compared to vehicle. Moreover, SVPs reduced myocardial scar, cardiomyocyte apoptosis and interstitial fibrosis, improved myocardial blood flow and neovascularization, and attenuated vascular permeability. SVPs secrete vascular endothelial growth factor A, angiopoietin-1, and chemokines and induce an endogenous angiocrine response by the host, through recruitment of vascular endothelial growth factor B expressing monocytes. The association of donor- and recipient-derived stimuli activates the proangiogenic and prosurvival Akt/eNOS/Bcl-2 signaling pathway. Moreover, microRNA-132 (miR-132) was constitutively expressed and secreted by SVPs and remarkably upregulated, together with its transcriptional activator cyclic AMP response element-binding protein, on stimulation by H/S or vascular endothelial growth factor B. We next investigated if SVP-secreted miR-132 acts as a paracrine activator of cardiac healing. In vitro studies showed that SVP conditioned medium stimulates endothelial tube formation and reduces myofibroblast differentiation, through inhibition of Ras-GTPase activating protein and methyl-CpG-binding protein 2, which are validated miR-132 targets. Furthermore, miR-132 inhibition by antimiR-132 decreased SVP capacity to improve contractility, reparative angiogenesis, and interstitial fibrosis in infarcted hearts. CONCLUSION: SVP transplantation produces long-term improvement of cardiac function through a novel paracrine mechanism involving the secretion of miR-132 and inhibition of its target genes.