Sathishkumar Kurusamy

Plasma Membrane Calcium ATPase Isoform 4 Inhibits Vascular Endothelial Growth Factor–Mediated Angiogenesis Through Interaction With Calcineurin

Objective— Vascular endothelial growth factor (VEGF) has been identified as a crucial regulator of physiological and pathological angiogenesis. Among the intracellular signaling pathways triggered by VEGF, activation of the calcineurin/nuclear factor of activated T cells (NFAT) signaling axis has emerged as a critical mediator of angiogenic processes. We and others previously reported a novel role for the plasma membrane calcium ATPase (PMCA) as an endogenous inhibitor of the calcineurin/NFAT pathway, via interaction with calcineurin, in cardiomyocytes and breast cancer cells. However, the functional significance of the PMCA/calcineurin interaction in endothelial pathophysiology has not been addressed thus far. Approach and Results— Using in vitro and in vivo assays, we here demonstrate that the interaction between PMCA4 and calcineurin in VEGF-stimulated endothelial cells leads to downregulation of the calcineurin/NFAT pathway and to a significant reduction in the subsequent expression of the NFAT-dependent, VEGF-activated, proangiogenic genes RCAN1.4 and Cox-2. PMCA4-dependent inhibition of calcineurin signaling translates into a reduction in endothelial cell motility and blood vessel formation that ultimately impairs in vivo angiogenesis by VEGF. Conclusions— Given the importance of the calcineurin/NFAT pathway in the regulation of pathological angiogenesis, targeted modulation of PMCA4 functionality might open novel therapeutic avenues to promote or attenuate new vessel formation in diseases that occur with angiogenesis.

Reduced expression of PMCA1 is associated with increased blood pressure with age which is preceded by remodelling of resistance arteries

Hypertension is a well‐established risk factor for adverse cardiovascular events, and older age is a risk factor for the development of hypertension. Genomewide association studies have linked ATP2B1, the gene for the plasma membrane calcium ATPase 1 (PMCA1), to blood pressure (BP) and hypertension. Here, we present the effects of reduction in the expression of PMCA1 on BP and small artery structure and function when combined with advancing age. Heterozygous PMCA1 null mice (PMCA1Ht) were generated and conscious BP was measured at 6 to 18 months of age. Passive and active properties of isolated small mesenteric arteries were examined by pressure myography. PMCA1Ht mice exhibited normal BP at 6 and 9 months of age but developed significantly elevated BP when compared to age‐matched wild‐type controls at ≥12 months of age. Decreased lumen diameter, increased wall thickness and increased wall:lumen ratio were observed in small mesenteric arteries from animals 9 months of age and older, indicative of eutrophic remodelling. Increases in mesenteric artery intrinsic tone and global intracellular calcium were evident in animals at both 6 and 18 months of age. Thus, decreased expression of PMCA1 is associated with increased BP when combined with advancing age. Changes in arterial structure precede the elevation of BP. Pathways involving PMCA1 may be a novel target for BP regulation in the elderly.

Poly-Gamma-Glutamic Acid (γ-PGA)-Based Encapsulation of Adenovirus to Evade Neutralizing Antibodies

In recent years, there has been an increasing interest in oncolytic adenoviral vectors as an alternative anticancer therapy. The induction of an immune response can be considered as a major limitation of this kind of application. Significant research efforts have been focused on the development of biodegradable polymer poly-gamma-glutamic acid (γ-PGA)-based nanoparticles used as a vector for effective and safe anticancer therapy, owing to their controlled and sustained-release properties, low toxicity, as well as biocompatibility with tissue and cells. This study aimed to introduce a specific destructive and antibody blind polymer-coated viral vector into cancer cells using γ-PGA and chitosan (CH). Adenovirus was successfully encapsulated into the biopolymer particles with an encapsulation efficiency of 92% and particle size of 485 nm using the ionic gelation method. Therapeutic agents or nanoparticles (NPs) that carry therapeutics can be directed specifically to cancerous cells by decorating their surfaces using targeting ligands. Moreover, in vitro neutralizing antibody response against viral capsid proteins can be somewhat reduced by encapsulating adenovirus into γ-PGA-CH NPs, as only 3.1% of the encapsulated adenovirus was detected by anti-adenovirus antibodies in the presented work compared to naked adenoviruses. The results obtained and the unique characteristics of the polymer established in this research could provide a reference for the coating and controlled release of viral vectors used in anticancer therapy.

Selective inhibition of plasma membrane calcium ATPase 4 improves angiogenesis and vascular reperfusion

AIMS Ischaemic cardiovascular disease is a major cause of morbidity and mortality worldwide. Despite promising results from pre-clinical animal models, VEGF-based strategies for therapeutic angiogenesis have yet to achieve successful reperfusion of ischaemic tissues in patients. Failure to restore efficient VEGF activity in the ischaemic organ remains a major problem in current pro-angiogenic therapeutic approaches. Plasma membrane calcium ATPase 4 (PMCA4) negatively regulates VEGF-activated angiogenesis via inhibition of the calcineurin/NFAT signalling pathway. PMCA4 activity is inhibited by the small molecule aurintricarboxylic acid (ATA). We hypothesize that inhibition of PMCA4 with ATA might enhance VEGF-induced angiogenesis. METHODS AND RESULTS We show that inhibition of PMCA4 with ATA in endothelial cells triggers a marked increase in VEGF-activated calcineurin/NFAT signalling that translates into a strong increase in endothelial cell motility and blood vessel formation. ATA enhances VEGF-induced calcineurin signalling by disrupting the interaction between PMCA4 and calcineurin at the endothelial-cell membrane. ATA concentrations at the nanomolar range, that efficiently inhibit PMCA4, had no deleterious effect on endothelial-cell viability or zebrafish embryonic development. However, high ATA concentrations at the micromolar level impaired endothelial cell viability and tubular morphogenesis, and were associated with toxicity in zebrafish embryos. In mice undergoing experimentally-induced hindlimb ischaemia, ATA treatment significantly increased the reperfusion of post-ischaemic limbs. CONCLUSIONS Our study provides evidence for the therapeutic potential of targeting PMCA4 to improve VEGF-based pro-angiogenic interventions. This goal will require the development of refined, highly selective versions of ATA, or the identification of novel PMCA4 inhibitors.

156 Inducers of pulmonary arterial hypertension upregulate the expression of plasma membrane calcium atpase 1 in pulmonary artery smooth muscle cells

Pulmonary arterial hypertension (PAH) is a chronic and life-threatening disease with high morbidity and mortality in adult and paediatric patients. PAH is characterised by a progressive narrowing and occlusion of small pulmonary arteries leading to increased pulmonary resistance, right ventricular hypertrophy, and, finally, right ventricular failure. A large body of data has shown that proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) represent key events in the vascular remodelling of pulmonary arteries that occurs during PAH. Levels of cytoplasmic calcium are an important determinant of PASMC proliferation and migration, and failure in maintaining appropriate levels of intracellular calcium are associated with PAH. The plasma membrane calcium ATPase (PMCA) proteins extrude calcium from the cytosol to the extracellular medium, and in doing so, play a critical role in the modulation of intracellular calcium levels. In this work, we have investigated whether inducers of PAH trigger any changes in the expression of PMCA proteins in PASMCs. Analysis of RNA expression levels for PMCA genes has revealed that treatment of PASMCs with PDGF results in a significant increase in the level of the RNA encoding for the protein PMCA1. Interestingly, PMCA1 RNA levels were also elevated in lungs of rats with monocrotaline-induced PAH. No changes were observed in the RNA levels for PMCA4, the other major PMCA isoform expressed in PASMCs. Although previous studies on the regulation of PMCA1 gene expression have identified functional binding sites for the transcription factors NFAT in the PMCA1 promoter region, we show here that PDGF-mediated upregulation of PMCA1 transcriptional expression is independent of activation of the calcineurin/NFAT signalling pathway. Our results suggest the involvement of PMCA1 in PASMC deregulation during PAH, although determination of the link between increased expression of PMCA1 and PAH requires further investigation.

144 Selective inhibition of plasma membrane calcium atpase 4 improves vegf-mediated angiogenesis

Ischaemic cardiovascular disease is the leading cause of death worldwide. Therapeutic angiogenesis aims to stimulate the growth of new blood vessels from pre-existing ones to reperfuse ischaemic tissues. Our laboratory is characterising the molecular mechanisms that regulate activation of the calcineurin/NFAT pathway during VEGF-induced angiogenesis. We recently showed that the Plasma Membrane Calcium ATPase 4 (PMCA4) negatively regulates angiogenesis by establishing a molecular interaction with calcineurin. The identification of aurintricarboxylic acid (ATA) as an inhibitor of PMCA4 prompted us to hypothesise that ATA will enhance VEGF-induced angiogenesis. Consistent with this hypothesis, we demonstrate in this work that inhibition of PMCA4 by treatment with ATA significantly increases the activity of calcineurin/NFAT signalling, and the subsequent expression of the NFAT-dependent, pro-angiogenic protein RCAN1.4 in VEGF-stimulated endothelial cells. Targeting PMCA4 with ATA significantly reduces the level of membrane-associated calcineurin, and the amount of calcineurin co-precipitated with PMCA4 in immunoprecipitation assays, indicating that ATA promotes disruption of the PMCA4/calcineurin interaction. ATA robustly enhances endothelial cell motility, and in vitro and in vivo blood vessel formation, with no harmful effects to the cells. Interestingly, incubation of HUVECs with low concentration of ATA had no effect on the viability of the cells or the development of zebra fish embryos. However, higher ATA concentrations were associated with cellular and embryo toxicity. Our study provides evidence for the therapeutic potential of targeting endothelial PMCA4 to improve VEGF-based pro-angiogenic interventions, and highlights possible clinical applications for PMCA4 inhibitors in the treatment of ischaemic cardiovascular disease.

P5 Pharmacological inhibition of plasma membrane calcium ATPASE 4 improves VEGF-induced angiogenesis

Ischaemic cardiovascular diseases are the leading cause of death worldwide and are often associated with partial or full occlusion of the blood vessel network in the affected organs. Therapeutic angiogenesis provides a valuable tool for treating cardiovascular diseases by stimulating the growth of new blood vessels from pre-existing ones. The pro-angiogenic factor Vascular Endothelial Growth Factor (VEGF) has been identified as a crucial regulator of angiogenesis through activation of the calcineurin/Nuclear Factor of Activated T-cells (NFAT) signalling pathway. We have previously reported a novel role for the Plasma Membrane Calcium ATPase 4 (PMCA4) as a negative regulator of angiogenesis via interaction with calcineurin. Aurintricarboxylic acid (ATA) has been recently identified as a PMCA4-specific inhibitor. We hypothesise that pharmacological inhibition of PMCA4 with ATA will enhance VEGF-mediated angiogenesis. Here, we show that treatment of endothelial cells with nanomolar concentrations of ATA notably enhances calcineurin/NFAT signalling, and the subsequent expression of the VEGF-induced, NFAT-dependent, pro-angiogenic protein RCAN1.4. Targeting PMCA4 with ATA reduces the level of membrane-associated calcineurin, and the amount of calcineurin co-precipitated with PMCA4 in immunoprecipitation assays, indicating that ATA promotes disruption of the PMCA4/calcineurin interaction. ATA mediated inhibition of PMCA4 also enhances endothelial cell motility, and both in vitro and in vivo blood vessel formation. Low concentrations of ATA do not have any deleterious effects on the viability of endothelial cells or the development of zebra fish embryos, highlighting the potential clinical use of ATA, at low concentrations, to improve blood vessel formation in patients with ischaemic cardiovascular diseases.

199 A Novel Selective Inhibitior for Plasma Membrane Calcium Atpase 4 Improves VEGF-Mediated Angiogenesis

Cardiovascular diseases such as ischaemic heart disease, peripheral arterial disease and stroke are leading causes of death worldwide. Therapeutic angiogenesis, which can improve the formation of new blood vessels in the ischaemic organ, provides a valuable tool for treating cardiovascular diseases. Angiogenesis, a complex blood vessel formation process, involves the participation of several pro- and anti-angiogenic factors. Among them, pro-angiogenic factor Vascular Endothelial Growth Factor (VEGF) has been identified to play a critical role in pathological angiogenesis. Pre-clinical studies demonstrate that VEGF-based pro-angiogenic therapies result in successful reperfusion of the ischaemic organ in animal models. In our previous study, we reported a novel role for the Plasma Membrane Calcium ATPase 4 (PMCA4) as a negative regulator of angiogenic processes mediated by VEGF. Here, we hypothesised that selective inhibition of PMCA4 with the small molecule aurintricarboxylic acid (ATA) will improve VEGF-driven angiogenesis in vitro and an in vivo model of mouse limb ischaemia. Consistent with this hypothesis, we demonstrate in this work that inhibition of PMCA4 by treatment with ATA significantly increases the activity of calcineurin/NFAT pathway and the subsequent expression of the NFAT-dependent, pro-angiogenic protein RCAN1.4 in VEGF-stimulated endothelial cells. Additionally, ATA treatment reduces the level of membrane-associated calcineurin, suggesting that enhancement of calcineurin signalling is the result of a disruption of the interaction between PMCA and calcineurin. Moreover, ATA treatment strongly enhances endothelial cell motility and capillary-like formation in matrigel assay. Furthermore, ATA significantly enhances MLEC motility in PMCA4 +/+ (wild type), but not in PMCA4 -/- (knock out) cells, conforming that ATA-mediated inhibition of PMCA4 is implicated in the increase of migration exerted by ATA. Importantly, long-term exposure of endothelial cells to ATA has no changes in cell viability, highlighting the potential clinical application of ATA. In this sense, Post-ischaemic reperfusion of ischaemic limbs in animals treated with ATA is significantly higher than in control-treated animals. The data from this study indicated that modulation of the activity of PMCA4 by treatment with ATA might have important clinical applications to promote blood vessel formation in human diseases associated with insufficient angiogenesis.

5 The cardiac microrna miR-1 induces endothelial cell angiogenesis

Ischaemic heart disease is the leading cause of morbidity and mortality in the Western world. Progression of the disease is associated with the growth and/or episodic rupture of atherosclerosis lesions leading to narrowing or occlusion of major coronary arteries. Arterial occlusion activates sprouting of pre-existing capillaries (angiogenesis) and the development of collateral arteries (arteriogenesis) to protect the myocardial tissue against the ischaemic condition. Unfortunately, the molecular mechanisms that control post-ischaemic neovascularization of the heart are large unknown. Emerging evidence indicates that acute ischaemia in patients with ST-segment-elevation myocardial infarction induces the release of microRNA miR-1 from injured cardiomyocytes. miRNA-1 promotes physiological angiogenesis during the embryogenesis of zebra fish but its role in post-natal angiogenesis in human endothelial cells has not been studied thus far. In this work, we show that adenoviral-mediated delivery of miR-1 into primary Human Umbilical Vein Endothelial Cells (HUVECs) resulted in an increase in proliferation, migration and the ability of the cells to form tubular-like structures in in vitro matrigel assays. These results suggest that cardiac microRNAs released by ischaemic cardiomyocytes might activate angiogenic events in neighbouring endothelial cells to promote the formation of new blood vessels. Thus, therapeutic interventions aimed to potentiate this process might have important clinical applications to facilitate fast perfusion of the infarcted heart. Acknowledgments This work was supported by RIHS, The Wolverhampton Coronary Aftercare Support Group (WCASG) Charity, and The Rotha Abraham Bequest Charity.

218 A Novel Role for the PMCA4-specific Inhibitor Aurintricarboxylic Acid as an Enhancer of VEGF-Induced Angiogenesis

Introduction Angiogenesis, the formation of new blood vessels from pre-existing ones, is a tightly regulated process essential for proper embryonic development, organ growth and tissue repair. Changes in the expression of the pro-angiogenic factor Vascular Endothelial Growth Factor (VEGF) have been reported to play a major role in the progression of several human diseases, such as diabetic retinopathy, tumour growth and acute limb ischemia, by altering normal vascularisation. VEGF binding to specific tyrosine kinase receptors located on the surface of endothelial cells activates a variety of signal transduction pathways that switch on the expression of specific target genes. Among them, VEGF-mediated activation of the calcineurin/NFAT signalling pathway has been identified as a crucial regulator of both physiological and pathological angiogenesis. Our laboratory is interested in the characterisation of the molecular mechanisms that regulate the activity of the calcineurin/NFAT pathway during VEGF-induced angiogenesis. In this sense, we have recently identified a novel role for the Plasma Membrane Calcium ATPase 4 protein as a negative regulator of angiogenesis via interaction with calcineurin. We hypothesise that inhibition of PMCA4 will promote angiogenesis, and thus, PMCA4 inhibition might be used to induce therapeutic angiogenesis. Methods To evaluate this hypothesis we have assayed VEGF-dependent proliferation, migration and tube formation in human primary endothelial cells treated with VEGF in the presence or absence of the PMCA4-specific inhibitor aurintricarboxylic acid (ATA). Results We show here that inhibition of PMCA4 by ATA significantly increased the VEGF-induced activation of the calcineurin/NFAT pathway, and the subsequent expression of the NFAT-dependent, pro-angiogenic protein RCAN1.4 in HUVEC endothelial cells. Furthermore, VEGF-triggered endothelial cell migration and tube formation was also enhanced by treatment of the cells with ATA. Interestingly, ATA had no effect on endothelial cell tubular morphogenesis in response to Fibroblast Growth Factor (a pro-angiogenic protein that induces angiogenesis in a calcineurin-independent manner). Long term incubation of endothelial cells with ATA did not alter the viability of the cells, highlighting its potential use in clinic. Examination of the effect of ATA on the activity of other endothelial molecules regulated by PMCA4 has shown that ATA reduces the phosphorylation of endothelial nitric oxide synthase (eNOS) in the regulatory residue Thr495, suggesting that the ATA-mediated effect on angiogenesis might be consequence of upregulation of several PMCA4-regulated signalling pathways. Conclusion Our results indicate that ATA might be used with therapeutic purposes to treat human diseases that occur with insufficient angiogenesis.