Roberto Berra-Romani

Ca2+ handling is altered when arterial myocytes progress from a contractile to a proliferative phenotype in culture.

Phenotypic modulation of vascular myocytes is important for vascular development and adaptation. A characteristic feature of this process is alteration in intracellular Ca(2+) handling, which is not completely understood. We studied mechanisms involved in functional changes of inositol 1,4,5-trisphosphate (IP(3))- and ryanodine (Ry)-sensitive Ca(2+) stores, store-operated Ca(2+) entry (SOCE), and receptor-operated Ca(2+) entry (ROCE) associated with arterial myocyte modulation from a contractile to a proliferative phenotype in culture. Proliferating, cultured myocytes from rat mesenteric artery have elevated resting cytosolic Ca(2+) levels and increased IP(3)-sensitive Ca(2+) store content. ATP- and cyclopiazonic acid [CPA; a sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor]-induced Ca(2+) transients in Ca(2+)-free medium are significantly larger in proliferating arterial smooth muscle cells (ASMCs) than in freshly dissociated myocytes, whereas caffeine (Caf)-induced Ca(2+) release is much smaller. Moreover, the Caf/Ry-sensitive store gradually loses sensitivity to Caf activation during cell culture. These changes can be explained by increased expression of all three IP(3) receptors and a switch from Ry receptor type II to type III expression during proliferation. SOCE, activated by depletion of the IP(3)/CPA-sensitive store, is greatly increased in proliferating ASMCs. Augmented SOCE and ROCE (activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol) in proliferating myocytes can be attributed to upregulated expression of, respectively, transient receptor potential proteins TRPC1/4/5 and TRPC3/6. Moreover, stromal interacting molecule 1 (STIM1) and Orai proteins are upregulated in proliferating cells. Increased expression of IP(3) receptors, SERCA2b, TRPCs, Orai(s), and STIM1 in proliferating ASMCs suggests that these proteins play a critical role in an altered Ca(2+) handling that occurs during vascular growth and remodeling.

Sodium pump α2 subunits control myogenic tone and blood pressure in mice

A key question in hypertension is: How is long‐term blood pressure controlled? A clue is that chronic salt retention elevates an endogenous ouabain‐like compound (EOLC) and induces salt‐dependent hypertension mediated by Na+/Ca2+ exchange (NCX). The precise mechanism, however, is unresolved. Here we study blood pressure and isolated small arteries of mice with reduced expression of Na+ pump α1 (α1+/−) or α2 (α2+/−) catalytic subunits. Both low‐dose ouabain (1–100 nm; inhibits only α2) and high‐dose ouabain (≥1 μm; inhibits α1) elevate myocyte Ca2+ and constrict arteries from α1+/−, as well as α2+/− and wild‐type mice. Nevertheless, only mice with reduced α2 Na+ pump activity (α2+/−), and not α1 (α1+/−), have elevated blood pressure. Also, isolated, pressurized arteries from α2+/−, but not α1+/−, have increased myogenic tone. Ouabain antagonists (PST 2238 and canrenone) and NCX blockers (SEA0400 and KB‐R7943) normalize myogenic tone in ouabain‐treated arteries. Only the NCX blockers normalize the elevated myogenic tone in α2+/− arteries because this tone is ouabain independent. All four agents are known to lower blood pressure in salt‐dependent and ouabain‐induced hypertension. Thus, chronically reduced α2 activity (α2+/− or chronic ouabain) apparently regulates myogenic tone and long‐term blood pressure whereas reduced α1 activity (α1+/−) plays no persistent role: the in vivo changes in blood pressure reflect the in vitro changes in myogenic tone. Accordingly, in salt‐dependent hypertension, EOLC probably increases vascular resistance and blood pressure by reducing α2 Na+ pump activity and promoting Ca2+ entry via NCX in myocytes.

Vascular Endothelial Growth Factor Stimulates Endothelial Colony Forming Cells Proliferation and Tubulogenesis by Inducing Oscillations in Intracellular Ca2+ Concentration†‡§

Endothelial progenitor cells (EPCs) home from the bone marrow to the site of tissue regeneration and sustain neovascularization after acute vascular injury and upon the angiogenic switch in solid tumors. Therefore, they represent a suitable tool for cell‐based therapy (CBT) in regenerative medicine and provide a novel promising target in the fight against cancer. Intracellular Ca2+ signals regulate numerous endothelial functions, such as proliferation and tubulogenesis. The growth of endothelial colony forming cells (ECFCs), which are EPCs capable of acquiring a mature endothelial phenotype, is governed by store‐dependent Ca2+ entry (SOCE). This study aimed at investigating the nature and the role of VEGF‐elicited Ca2+ signals in ECFCs. VEGF induced asynchronous Ca2+ oscillations, whose latency, amplitude, and frequency were correlated to the growth factor dose. Removal of external Ca2+ (0Ca2+) and SOCE inhibition with N‐(4‐[3,5‐bis(trifluoromethyl)‐1H‐pyrazol‐1‐yl]phenyl)‐4‐methyl‐1,2,3‐thiadiazole‐5‐carboxamide (BTP‐2) reduced the duration of the oscillatory signal. Blockade of phospholipase C‐γ with U73122, emptying the inositol‐1,4,5‐trisphosphate (InsP3)‐sensitive Ca2+ pools with cyclopiazonic acid (CPA), and inhibition of InsP3 receptors with 2‐APB prevented the Ca2+ response to VEGF. VEGF‐induced ECFC proliferation and tubulogenesis were inhibited by the Ca2+‐chelant, BAPTA, and BTP‐2. NF‐κB activation by VEGF was impaired by BAPTA, BTP‐2, and its selective blocker, thymoquinone. Thymoquinone, in turn, suppressed VEGF‐dependent ECFC proliferation and tubulogenesis. These data indicate that VEGF‐induced Ca2+ oscillations require the interplay between InsP3‐dependent Ca2+ release and SOCE, and promote ECFC growth and tubulogenesis by engaging NF‐κB. This novel signaling pathway might be exploited to enhance the outcome of CBT and chemotherapy. STEM CELLS 2011;29:1898–1907

Upregulation of Na+ and Ca2+ transporters in arterial smooth muscle from ouabain-induced hypertensive rats

Prolonged ouabain administration (25 microg kg(-1) day(-1) for 5 wk) induces ouabain hypertension (OH) in rats, but the molecular mechanisms by which ouabain elevates blood pressure are unknown. Here, we compared Ca(2+) signaling in mesenteric artery smooth muscle cells (ASMCs) from normotensive (NT) and OH rats. Resting cytosolic free Ca(2+) concentration ([Ca(2+)](cyt); measured with fura-2) and phenylephrine-induced Ca(2+) transients were augmented in freshly dissociated OH ASMCs. Immunoblots revealed that the expression of the ouabain-sensitive alpha(2)-subunit of Na(+) pumps, but not the predominant, ouabain-resistant alpha(1)-subunit, was increased (2.5-fold vs. NT ASMCs) as was Na(+)/Ca(2+) exchanger-1 (NCX1; 6-fold vs. NT) in OH arteries. Ca(2+) entry, activated by sarcoplasmic reticulum (SR) Ca(2+) store depletion with cyclopiazonic acid (SR Ca(2+)-ATPase inhibitor) or caffeine, was augmented in OH ASMCs. This reflected an augmented expression of 2.5-fold in OH ASMCs of C-type transient receptor potential TRPC1, an essential component of store-operated channels (SOCs); two other components of some SOCs were not expressed (TRPC4) or were not upregulated (TRPC5). Ba(2+) entry activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol [a measure of receptor-operated channel (ROC) activity] was much greater in OH than NT ASMCs. This correlated with a sixfold upregulation of TRPC6 protein, a ROC family member. Importantly, in primary cultured mesenteric ASMCs from normal rats, 72-h treatment with 100 nM ouabain significantly augmented NCX1 and TRPC6 protein expression and increased resting [Ca(2+)](cyt) and ROC activity. SOC activity was also increased. Silencer RNA knockdown of NCX1 markedly downregulated TRPC6 and eliminated the ouabain-induced augmentation; silencer RNA knockdown of TRPC6 did not affect NCX1 expression but greatly attenuated its upregulation by ouabain. Clearly, NCX1 and TRPC6 expression are interrelated. Thus, prolonged ouabain treatment upregulates the Na(+) pump alpha(2)-subunit-NCX1-TRPC6 (ROC) Ca(2+) signaling pathway in arterial myocytes in vitro as well as in vivo. This may explain the augmented myogenic responses and enhanced phenylephrine-induced vasoconstriction in OH arteries (83) as well as the high blood pressure in OH rats.

Update on vascular endothelial Ca(2+) signalling: A tale of ion channels, pumps and transporters.

A monolayer of endothelial cells (ECs) lines the lumen of blood vessels and forms a multifunctional transducing organ that mediates a plethora of cardiovascular processes. The activation of ECs from as state of quiescence is, therefore, regarded among the early events leading to the onset and progression of potentially lethal diseases, such as hypertension, myocardial infarction, brain stroke, and tumor. Intracellular Ca(2+) signals have long been know to play a central role in the complex network of signaling pathways regulating the endothelial functions. Notably, recent work has outlined how any change in the pattern of expression of endothelial channels, transporters and pumps involved in the modulation of intracellular Ca(2+) levels may dramatically affect whole body homeostasis. Vascular ECs may react to both mechanical and chemical stimuli by generating a variety of intracellular Ca(2+) signals, ranging from brief, localized Ca(2+) pulses to prolonged Ca(2+) oscillations engulfing the whole cytoplasm. The well-defined spatiotemporal profile of the subcellular Ca(2+) signals elicited in ECs by specific extracellular inputs depends on the interaction between Ca(2+) releasing channels, which are located both on the plasma membrane and in a number of intracellular organelles, and Ca(2+) removing systems. The present article aims to summarize both the past and recent literature in the field to provide a clear-cut picture of our current knowledge on the molecular nature and the role played by the components of the Ca(2+) machinery in vascular ECs under both physiological and pathological conditions.

Epidermal growth factor induces intracellular Ca2+ oscillations in microvascular endothelial cells

An increase in intracellular Ca2+ concentration ([Ca2+]i) may play a role in the proliferative effect of several growth factors. In this study, the changes in [Ca2+]i elicited by epidermal growth factor (EGF) in rat cardiac microvascular endothelial cells (CMEC) have been investigated by using fura‐2 conventional and confocal microscopy. A large heterogeneity in the latency and in the pattern of the Ca2+ response was found at each dose of EGF (2.5–100 ng/ml), whereas some cells displayed a non‐oscillatory behavior and others exhibited a variable number of Ca2+ oscillations. On average, the fraction of responsive cells, the total number of oscillations and the duration of the Ca2+ signal were higher at around 10 ng/ml EGF, while there was no dose‐dependence in the lag time and in the amplitude of the [Ca2+]i increase. EGF‐induced Ca2+ spikes were abolished by the tyrosine kinase inhibitor genistein, but not by its inactive analogue daidzein, and by the phospholipase C blocker NCDC. Only 1–2 transients could be elicited in Ca2+‐free solution, while re‐addition of extracellular Ca2+ recovered the spiking activity. The oscillatory signal was prevented by the SERCA inhibitor thapsigargin and abolished by the calcium entry blockers Ni2+ and La3+. Moreover, EGF‐induced Ca2+ transients were abolished by the InsP3 receptor blocker caffeine, while ryanodine was without effect. Confocal imaging microscopy showed that the Ca2+ response to EGF was localized both in the cytoplasm and in the nucleus. We suggest that EGF‐induced [Ca2+]i increase may play a role in the proliferative action of EGF on endothelial cells.

Ca2+-dependent nitric oxide release in the injured endothelium of excised rat aorta: a promising mechanism applying in vascular prosthetic devices in aging patients

BackgroundNitric oxide is key to endothelial regeneration, but it is still unknown whether endothelial cell (EC) loss results in an increase in NO levels at the wound edge. We have already shown that endothelial damage induces a long-lasting Ca2+ entry into surviving cells though connexin hemichannels (CxHcs) uncoupled from their counterparts on ruptured cells. The physiological outcome of injury-induced Ca2+ inflow is, however, unknown.MethodsIn this study, we sought to determine whether and how endothelial scraping induces NO production (NOP) in the endothelium of excised rat aorta by exploiting the NO-sensitive fluorochrome, DAF-FM diacetate and the Ca2+-sensitive fluorescent dye, Fura-2/AM.ResultsWe demonstrated that injury-induced NOP at the lesion site is prevented in presence of the endothelial NO synthase inhibitor, L-NAME, and in absence of extracellular Ca2+. Unlike ATP-dependent NO liberation, the NO response to injury is insensitive to BTP-2, which selectively blocks store-operated Ca2+ inflow. However, injury-induced NOP is significantly reduced by classic gap junction blockers, and by connexin mimetic peptides specifically targeting Cx37Hcs, Cx40HCs, and Cx43Hcs. Moreover, disruption of caveolar integrity prevents injury-elicited NO signaling, but not the accompanying Ca2+ response.ConclusionsThe data presented provide the first evidence that endothelial scraping stimulates NO synthesis at the wound edge, which might both exert an immediate anti-thrombotic and anti-inflammatory action and promote the subsequent re-endothelialization.

Low-dose ouabain constricts small arteries from ouabain-hypertensive rats: implications for sustained elevation of vascular resistance.

Prolonged ouabain administration to normal rats causes sustained blood pressure (BP) elevation. This ouabain-induced hypertension (OH) has been attributed, in part, to the narrowing of third-order resistance arteries (approximately 320 microm internal diameter) as a result of collagen deposition in the artery media. Here we describe the structural and functional properties of fourth-order mesenteric small arteries from control and OH rats, including the effect of low-dose ouabain on myogenic tone in these arteries. Systolic BP in OH rats was 138 +/- 3 versus 124 +/- 4 mmHg in controls (P < 0.01). Pressurized (70 mmHg) control and OH arteries, with only a single layer of myocytes, both had approximately 165-microm internal diameters and approximately 20-microm wall thicknesses. Even after fixation, despite vasoconstriction, the diameters and wall thicknesses did not differ between control and OH fourth-order arteries, whereas in third-order arteries, both parameters were significantly smaller in OH than in controls. Myogenic reactivity was significantly augmented in OH fourth-order arteries. Nevertheless, phenylephrine- (1 microM) and high K(+)-induced vasoconstrictions and acetylcholine-induced vasodilation were comparable in control and OH arteries. Vasoconstrictions induced by 5 microM phenylephrine and by 10 mM caffeine in Ca(2+)-free media indicated that releasable sarcoplasmic reticulum Ca(2+) stores were normal in OH arteries. Importantly, 100 nM ouabain constricted both control and OH arteries by approximately 26 microm, indicating that this response was not downregulated in OH rats. This maximal ouabain-induced constriction corresponds to a approximately 90% increase in resistance to flow in these small arteries; thus ouabain at EC(50) of approximately 0.66 nM should raise resistance by approximately 35%. We conclude that dynamic constriction in response to circulating nanomolar ouabain in small arteries likely makes a major contribution to the increased vascular tone and BP in OH rats.

The Mechanism of Injury-Induced Intracellular Calcium Concentration Oscillations in the Endothelium of Excised Rat Aorta

Endothelial injury is the primary event that leads to a variety of severe vascular disorders. Mechanical injury elicits a Ca²⁺ response in the endothelium of excised rat aorta, which comprises an initial Ca²⁺ release from inositol-1,4,5-trisphosphate (InsP₃)-sensitive stores followed by a long-lasting decay phase due to Ca²⁺ entry through uncoupled connexons. The Ca²⁺ signal may also adopt an oscillatory pattern, the molecular underpinnings of which are unclear. In the light of the role played by Ca²⁺ spiking in tissue regeneration, this study aimed to unveil the mechanisms underlying injury-induced Ca²⁺ oscillations. The latter reversibly ceased upon removal of extracellular Ca²⁺ or addition of the gap junction blockers heptanol, 18 α,β-glycyrrhetinic acid, La³⁺ and Ni²⁺, but were insensitive to BTP-2 and SKF 96365. The spiking response was abolished by inhibiting the Ca²⁺ entry mode of the Na⁺/Ca²⁺ exchanger (NCX). The InsP₃-producing agonist ATP resumed Ca²⁺ oscillations in silent cells, while the phospholipase C inhibitor U73122 suppressed them. Injury-induced Ca²⁺ transients were prevented by the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) blockers thapsigargin and cyclopiazonic acid, while they were unaffected by suramin and genistein. These data show for the first time that the coordinated interplay between NCX-mediated Ca²⁺ entry and InsP₃-dependent Ca²⁺ release contributes to injury-induced intracellular Ca²⁺ concentration oscillations.

Ca2+ signaling in injured in situ endothelium of rat aorta

The inner wall of excised rat aorta was scraped by a microelectrode and Ca2+ signals were investigated by fluorescence microscopy in endothelial cells (ECs) directly coupled with injured cells. The injury caused an immediate increase in the intracellular Ca2+ concentration ([Ca2+]i), followed by a long-lasting decay phase due to Ca2+ influx from extracellular space. The immediate response was mainly due to activation of purinergic receptors, as shown by the effect of P2X and P2Y receptors agonists and antagonists, such as suramin, alpha,beta-MeATP, MRS-2179 and 2-MeSAMP. Inhibition of store-operated Ca2+ influx did not affect either the peak response or the decay phase. Furthermore, the latter was: (i) insensitive to phospholipase C inhibition, (ii) sensitive to the gap junction blockers, palmitoleic acid, heptanol, octanol and oleamide, and (iii) sensitive to La3+ and Ni2+, but not to Gd3+. Finally, ethidium bromide or Lucifer Yellow did not enter ECs facing the scraped area. These results suggest that endothelium scraping: (i) causes a short-lasting stimulation of healthy ECs by extracellular nucleotides released from damaged cells and (ii) uncouples the hemichannels of the ECs facing the injury site; these hemichannels do not fully close and allow a long-lasting Ca2+ entry.