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Emerging evidence indicates that short (~22 nucleotides) non-coding RNA molecules called microRNAs (miRNAs) play a key role in the regulation of post-ischaemic angiogenesis. miRNAs control gene expression by binding to complementary sequences of specific mRNA transcripts, leading to degradation or translational repression of the targeted mRNA. miR-133a is highly expressed and plays a crucial role in skeletal and cardiac muscle biology. The expression level of miR-133a in vascular endothelial cells is very low under physiological conditions. However, multiple cardiovascular risk factors including oxidized-LDL, pro-inflammatory cytokines, hyperglycemia, dyslipidemia, and hyperhomocysteinemia induce aberrant miR-133a expression in endothelial cells leading to endothelial dysfunction. Endothelial dysfunction in cardiovascular ischaemic disease is often accompanied by impairment of reparative angiogenic processes. Here, we have evaluated the role of aberrant miR133a expression in VEGF-mediated angiogenesis. We show that exogenous expression of miR-133a in Human Umbilical Vein Endothelial Cells (HUVEC) significantly reduces VEGF-induced endothelial cell proliferation. Moreover, adenoviral-mediated delivery of cardiac miR133a into endothelial cells inhibits HUVEC cell motility and tubular morphogenesis. Interestingly, downregulation of phosphorylation (activation) of the signalling protein Erk1/2 by miR-133a was detected in VEGF stimulated HUVEC cells. Our results demonstrate that aberrant expression of miR133a has an anti-angiogenic effect in endothelial cells, and strongly suggest that targeted strategies to suppress ectopic expression of miR-133a in the dysfunctional endothelium might have important therapeutic applications to improve reparative angiogenic process in patients suffering from ischaemic cardiovascular disease. Acknowledgement This work was supported by the Rotha Abraham Bequest Charity

Pulmonary arterial hypertension (PAH) is a chronic and life-threatening disease characterised by a progressive narrowing and occlusion of small pulmonary arteries leading to increased pulmonary resistance, right ventricular hypertrophy, and, finally, right ventricular failure. Several studies have demonstrated that proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) play a pivotal role in the vascular remodelling characteristic of PAH. Levels of cytoplasmic calcium are an important determinant of PASMC proliferation and migration. The Plasma Membrane Calcium ATPase (PMCA) proteins extrude calcium from the cytosol to the extracellular medium. Here, we have investigated whether inducers of PAH trigger any changes in the expression of PMCA genes in PASMCs. Treatment of PASMCs with the PAH inducers Platelet Derived Growth Factor (PDGF) or TNF-alpha induced a significant increase in the RNA levels of PMCA1. PMCA1 RNA levels are also elevated in lungs of rats with monocrotaline-induced PAH. In silico analysis of the PMCA1 gene promoter region has shown putative binding sites for the transcription factors NFAT and NFkB that could mediate the transcriptional upregulation triggered by PDGF and TNF-alpha respectively. However, we show here that upregulated PMCA1 gene expression is not mediated by binding of these transcription factors to the proximal promoter region. No changes were observed in the RNA levels of PMCA4, the other major PMCA isoform expressed in PASMCs. Our results suggest an important role for PMCA1 in PASMC deregulation during PAH, although a full understanding of the role of PMCA1 on the onset and progression of PAH requires further investigation.

Angiogenesis requires co-ordination of multiple signalling inputs to regulate the behaviour of endothelial cells (ECs) as they form vascular networks. Vascular endothelial growth factor (VEGF) is essential for angiogenesis and induces downstream signalling pathways including increased cytosolic calcium levels. Here we show that transmembrane protein 33 (tmem33), which has no known function in multicellular organisms, is essential to mediate effects of VEGF in both zebrafish and human ECs. We find that tmem33 localises to the endoplasmic reticulum in zebrafish ECs and is required for cytosolic calcium oscillations in response to Vegfa. tmem33-mediated endothelial calcium oscillations are critical for formation of endothelial tip cell filopodia and EC migration. Global or endothelial-cell-specific knockdown of tmem33 impairs multiple downstream effects of VEGF including ERK phosphorylation, Notch signalling and embryonic vascular development. These studies reveal a hitherto unsuspected role for tmem33 and calcium oscillations in the regulation of vascular development., Calcium signalling downstream of VEGF is essential for VEGF-induced angiogenesis. Here Savage et al. show that Transmembrane Protein 33 (TMEM33) is required for angiogenesis and the endothelial calcium response to VEGF, revealing a function for TMEM33 in multicellular organisms.

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.

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.

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.

Current anti-angiogenic treatments involve the attenuation of signalling via the pro-angiogenic vascular endothelial growth factor/receptor (VEGF/VEGFR) axis. Stimulation of angiogenesis by VEGF requires the activation of the calcineurin/nuclear factor of activated T-cells (NFAT) signal transduction pathway which is inhibited by Plasma Membrane Calcium ATPase 4 (PMCA4), an endogenous calcium extrusion pump. However, PMCA4s role in calcineurin/NFAT-dependent angiogenesis is unknown. Using “gain of function” studies, we show here that adenoviral overexpression of PMCA4 in human umbilical vein endothelial cells (HUVEC) inhibited NFAT activity, decreased the expression of NFAT-dependent pro-angiogenic proteins (regulator of calcineurin 1.4 (RCAN1.4) and cyclooxygenase-2) and diminished in vitro cell migration and tube formation in response to VEGF-stimulation. Furthermore, in vivo blood vessel formation was attenuated in a matrigel plug assay by ectopic expression of PMCA4. Conversely, “loss of function” experiments by si-RNA-mediated knockdown of PMCA4 in HUVEC or isolation of mouse lung endothelial cells from PMCA4−/− mice showed increased VEGF-induced NFAT activity, RCAN1.4 expression, in vitro endothelial cell migration, tube formation and in vivo blood vessel formation. Additionally, in an in vivo pathological angiogenesis model of limb ischemia, the reperfusion of the ischemic limb of PMCA4−/− mice was augmented compared to wild-type. Disruption of the interaction between endogenous PMCA4 and calcineurin by adenoviral overexpression of the region of PMCA4 that interacts with calcineurin (residues 428–651) increased NFAT activity, RCAN1.4 protein expression and in vitro tube formation. These results identify PMCA4 as an inhibitor of VEGF-induced angiogenesis, highlighting its potential as a new therapeutic target for anti-angiogenic treatments.

Angiogenesis, the formation of new capillaries from pre-existing ones, is a biological process essential for successful embryonic development, organ growth and tissue repair. Excessive or insufficient growth of blood vessels, due to de-regulation of this process, plays a major role in the pathophysiology of several human diseases. The pro-angiogenic factor Vascular Endothelial Growth Factor (VEGF) has been identified as a crucial regulator of both physiological and pathological angiogenesis. VEGF activates the calcineurin/Nuclear Factor of Activated T-cells (NFAT) signalling pathway which is a critical mediator of angiogenesis. Our group has recently identified a novel role for the plasma membrane calcium ATPase 4 (PMCA4) as a negative regulator of VEGF-dependent angiogenesis. The small molecule Aurintricarboxylic acid (ATA) has been characterised as a selective inhibitor of PMCA4. We hypothesise that ATA-mediated inhibition of PMCA4 in endothelial cells will enhance VEGF-driven angiogenesis. Consistent with this hypothesis, we demonstrate in this work that inhibition of PMCA4 by treatment with ATA increases calcineurin/NFAT activity and the expression of the NFAT-dependent, pro-angiogenic protein RCAN1.4 in VEGF-stimulated HUVEC. Moreover, ATA treatment significantly enhanced endothelial cell migration and tubular morphogenesis in response to VEGF-stimulation. Interestingly, incubation of HUVEC with ATA had no effect on the cell viability or Erk1/2 phosphorylation (activation) status of VEGF-stimulated cells. Our results show a novel role for the PMCA4 specific inhibitor ATA as a stimulator of VEGF-induced angiogenesis. Thus, ATA might lead to the design of new therapeutic strategies to improve blood vessel formation in diseases associated with insufficient angiogenesis.