Robert Little

Plasma membrane calcium ATPases (PMCAs) as potential targets for the treatment of essential hypertension

The incidence of hypertension, the major modifiable risk factor for cardiovascular disease, is increasing. Thus, there is a pressing need for the development of new and more effective strategies to prevent and treat hypertension. Development of these relies on a continued evolution of our understanding of the mechanisms which control blood pressure (BP). Resistance arteries are important in the regulation of total peripheral resistance and BP; changes in their structure and function are strongly associated with hypertension. Anti-hypertensives which both reduce BP and reverse changes in resistance arterial structure reduce cardiovascular risk more than therapies which reduce BP alone. Hence, identification of novel potential vascular targets which modify BP is important. Hypertension is a multifactorial disorder which may include a genetic component. Genome wide association studies have identified ATP2B1, encoding the calcium pump plasma membrane calcium ATPase 1 (PMCA1), as having a strong association with BP and hypertension. Knockdown or reduced PMCA1 expression in mice has confirmed a physiological role for PMCA1 in BP and resistance arterial regulation. Altered expression or inhibition of PMCA4 has also been shown to modulate these parameters. The mechanisms whereby PMCA1 and 4 can modulate vascular function remain to be fully elucidated but may involve regulation of intracellular calcium homeostasis and/or comprise a structural role. However, clear physiological links between PMCA and BP, coupled with experimental studies directly linking PMCA1 and 4 to changes in BP and arterial function, suggest that they may be important targets for the development of new pharmacological modulators of BP.

Calcium Extrusion Pump PMCA4: A New Player in Renal Calcium Handling?

Calcium (Ca2+) is vital for multiple processes in the body, and maintenance of the electrolyte concentration is required for everyday physiological function. In the kidney, and more specifically, in the late distal convoluted tubule and connecting tubule, the fine-tuning of Ca2+ reabsorption from the pro-urine takes place. Here, Ca2+ enters the epithelial cell via the transient receptor potential vanilloid receptor type 5 (TRPV5) channel, diffuses to the basolateral side bound to calbindin-D28k and is extruded to the blood compartment via the Na+/Ca2+ exchanger 1 (NCX1) and the plasma membrane Ca2+ ATPase (PMCA). Traditionally, PMCA1 was considered to be the primary Ca2+ pump in this process. However, in recent studies TRPV5-expressing tubules were shown to highly express PMCA4. Therefore, PMCA4 may have a predominant role in renal Ca2+ handling. This study aimed to elucidate the role of PMCA4 in Ca2+ homeostasis by characterizing the Ca2+ balance, and renal and duodenal Ca2+-related gene expression in PMCA4 knockout mice. The daily water intake of PMCA4 knockout mice was significantly lower compared to wild type littermates. There was no significant difference in serum Ca2+ level or urinary Ca2+ excretion between groups. In addition, renal and duodenal mRNA expression levels of Ca2+-related genes, including TRPV5, TRPV6, calbindin-D28k, calbindin-D9k, NCX1 and PMCA1 were similar in wild type and knockout mice. Serum FGF23 levels were significantly increased in PMCA4 knockout mice. In conclusion, PMCA4 has no discernible role in normal renal Ca2+ handling as no urinary Ca2+ wasting was observed. Further investigation of the exact role of PMCA4 in the distal convoluted tubule and connecting tubule is required.

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.

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.

Acute inhibition of PMCA4, but not global ablation, reduces blood pressure and arterial contractility via a nNOS-dependent mechanism

Cardiovascular disease is the worlds leading cause of morbidity and mortality, with high blood pressure (BP) contributing to increased severity and number of adverse outcomes. Plasma membrane calcium ATPase 4 (PMCA4) has been previously shown to modulate systemic BP. However, published data are conflicting, with both overexpression and inhibition of PMCA4 in vivo shown to increase arterial contractility. Hence, our objective was to determine the role of PMCA4 in the regulation of BP and to further understand how PMCA4 functionally regulates BP using a novel specific inhibitor to PMCA4, aurintricarboxylic acid (ATA). Our approach assessed conscious BP and contractility of resistance arteries from PMCA4 global knockout (PMCA4KO) mice compared to wild‐type animals. Global ablation of PMCA4 had no significant effect on BP, arterial structure or isolated arterial contractility. ATA treatment significantly reduced BP and arterial contractility in wild‐type mice but had no significant effect in PMCA4KO mice. The effect of ATAin vivo and ex vivo was abolished by the neuronal nitric oxide synthase (nNOS) inhibitor Vinyl‐l‐NIO. Thus, this highlights differences in the effects of PMCA4 ablation and acute inhibition on the vasculature. Importantly, for doses here used, we show the vascular effects of ATA to be specific for PMCA4 and that ATA may be a further experimental tool for elucidating the role of PMCA4.

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.

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.