Ivica Grgic

Evidence for a Functional Role of Endothelial Transient Receptor Potential V4 in Shear Stress–Induced Vasodilatation

Objective—Ca2+-influx through transient receptor potential (TRP) channels was proposed to be important in endothelial function, although the precise role of specific TRP channels is unknown. Here, we investigated the role of the putatively mechanosensitive TRPV4 channel in the mechanisms of endothelium-dependent vasodilatation. Methods and Results—Expression and function of TRPV4 was investigated in rat carotid artery endothelial cells (RCAECs) by using in situ patch-clamp techniques, single-cell RT-PCR, Ca2+ measurements, and pressure myography in carotid artery (CA) and Arteria gracilis. In RCAECs in situ, TRPV4 currents were activated by the selective TRPV4 opener 4&agr;-phorbol-12,13-didecanoate (4&agr;PDD), arachidonic acid, moderate warmth, and mechanically by hypotonic cell swelling. Single-cell RT-PCR in endothelial cells demonstrated mRNA expression of TRPV4. In FURA-2 Ca2+ measurements, 4&agr;PDD increased [Ca2+]i by ≈140 nmol/L above basal levels. In pressure myograph experiments in CAs and A gracilis, 4&agr;PDD caused robust endothelium-dependent and strictly endothelium-dependent vasodilatations by ≈80% (KD 0.3 &mgr;mol/L), which were suppressed by the TRPV4 blocker ruthenium red (RuR). Shear stress–induced vasodilatation was similarly blocked by RuR and also by the phospholipase A2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF3). 4&agr;PDD produced endothelium-derived hyperpolarizing factor (EDHF)–type responses in A gracilis but not in rat carotid artery. Shear stress did not produce EDHF-type vasodilatation in either vessel type. Conclusions—Ca2+ entry through endothelial TRPV4 channels triggers NO- and EDHF-dependent vasodilatation. Moreover, TRPV4 appears to be mechanistically important in endothelial mechanosensing of shear stress.

Blockade of the Intermediate-Conductance Calcium-Activated Potassium Channel as a New Therapeutic Strategy for Restenosis

Background—Angioplasty stimulates proliferation and migration of vascular smooth muscle cells (VSMC), leading to neointimal thickening and vascular restenosis. In a rat model of balloon catheter injury (BCI), we investigated whether alterations in expression of Ca2+-activated K+ channels (KCa) contribute to intimal hyperplasia and vascular restenosis. Methods and Results—Function and expression of KCa in mature medial and neointimal VSMC were characterized in situ by combined single-cell RT-PCR and patch-clamp analysis. Mature medial VSMC exclusively expressed large-conductance KCa (BKCa) channels. Two weeks after BCI, expression of BKCa was significantly reduced in neointimal VSMC, whereas expression of intermediate-conductance KCa (IKCa1) channels was upregulated. In the aortic VSMC cell line, A7r5 epidermal growth factor (EGF) induced IKCa1 upregulation and EGF-stimulated proliferation was suppressed by the selective IKCa1 blocker TRAM-34. Daily in vivo administration of TRAM-34 to rats significantly reduced intimal hyperplasia by ≈40% at 1, 2, and 6 weeks after BCI. Two weeks of treatment with the related compound clotrimazole was equally effective. Reduction of intimal hyperplasia was accompanied by decreased neointimal cell content, with no change in the rate of apoptosis or collagen content. Conclusions—The switch toward IKCa1 expression may promote excessive neointimal VSMC proliferation. Blockade of IKCa1 could therefore represent a new therapeutic strategy to prevent restenosis after angioplasty.

Impaired Endothelium-Derived Hyperpolarizing Factor-Mediated Dilations and Increased Blood Pressure in Mice Deficient of the Intermediate-Conductance Ca2+-Activated K+ Channel

The endothelium plays a key role in the control of vascular tone and alteration in endothelial cell function contributes to several cardiovascular disease states. Endothelium-dependent dilation is mediated by NO, prostacyclin, and an endothelium-derived hyperpolarizing factor (EDHF). EDHF signaling is thought to be initiated by activation of endothelial Ca2+-activated K+ channels (KCa), leading to hyperpolarization of the endothelium and subsequently to hyperpolarization and relaxation of vascular smooth muscle. In the present study, we tested the functional role of the endothelial intermediate-conductance KCa (IKCa/KCa3.1) in endothelial hyperpolarization, in EDHF-mediated dilation, and in the control of arterial pressure by targeted deletion of KCa3.1. KCa3.1-deficient mice (KCa3.1−/−) were generated by conventional gene-targeting strategies. Endothelial KCa currents and EDHF-mediated dilations were characterized by patch-clamp analysis, myography and intravital microscopy. Disruption of the KCa3.1 gene abolished endothelial KCa3.1 currents and significantly diminished overall current through KCa channels. As a consequence, endothelial and smooth muscle hyperpolarization in response to acetylcholine was reduced in KCa3.1−/− mice. Acetylcholine-induced dilations were impaired in the carotid artery and in resistance vessels because of a substantial reduction of EDHF-mediated dilation in KCa3.1−/− mice. Moreover, the loss of KCa3.1 led to a significant increase in arterial blood pressure and to mild left ventricular hypertrophy. These results indicate that the endothelial KCa3.1 is a fundamental determinant of endothelial hyperpolarization and EDHF signaling and, thereby, a crucial determinant in the control of vascular tone and overall circulatory regulation.

Targeted proximal tubule injury triggers interstitial fibrosis and glomerulosclerosis

Chronic kidney disease (CKD) remains one of the leading causes of death in the developed world and acute kidney injury (AKI) is now recognized as a major risk factor in its development. Understanding the factors leading to CKD after acute injury are limited by current animal models of AKI which concurrently target various kidney cell types such as epithelial, endothelial and inflammatory cells. Here we developed a mouse model of kidney injury using the Six2-Cre-LoxP technology to selectively activate expression of the simian diphtheria toxin receptor in renal epithelia derived from the metanephric mesenchyme. By adjusting the timing and dose of diphtheria toxin a highly selective model of tubular injury was created to define the acute and chronic consequences of isolated epithelial injury. The diphtheria toxin-induced sublethal tubular epithelial injury was confined to the S1 and S2 segments of the proximal tubule rather than being widespread in the metanephric mesenchyme derived epithelial lineage. Acute injury was promptly followed by inflammatory cell infiltration and robust tubular cell proliferation leading to complete recovery after a single toxin insult. In striking contrast, three insults to renal epithelial cells at one week intervals resulted in maladaptive repair with interstitial capillary loss, fibrosis and glomerulosclerosis which was highly correlated with the degree of interstitial fibrosis. Thus, selective epithelial injury can drive the formation of interstitial fibrosis, capillary rarefaction and potentially glomerulosclerosis, substantiating a direct role for damaged tubule epithelium in the pathogenesis of CKD.

Genetic deficit of SK3 and IK1 channels disrupts the endothelium-derived hyperpolarizing factor vasodilator pathway and causes hypertension.

Background— It has been proposed that activation of endothelial SK3 (KCa2.3) and IK1 (KCa3.1) K+ channels plays a role in the arteriolar dilation attributed to an endothelium-derived hyperpolarizing factor (EDHF). However, our understanding of the precise function of SK3 and IK1 in the EDHF dilator response and in blood pressure control remains incomplete. To clarify the roles of SK3 and IK1 channels in the EDHF dilator response and their contribution to blood pressure control in vivo, we generated mice deficient for both channels. Methods and Results— Expression and function of endothelial SK3 and IK1 in IK1−/−/SK3T/T mice was characterized by patch-clamp, membrane potential measurements, pressure myography, and intravital microscopy. Blood pressure was measured in conscious mice by telemetry. Combined IK1/SK3 deficiency in IK1−/−/SK3T/T (+doxycycline) mice abolished endothelial KCa currents and impaired acetylcholine-induced smooth muscle hyperpolarization and EDHF-mediated dilation in conduit arteries and in resistance arterioles in vivo. IK1 deficiency had a severe impact on acetylcholine-induced EDHF-mediated vasodilation, whereas SK3 deficiency impaired NO-mediated dilation to acetylcholine and to shear stress stimulation. As a consequence, SK3/IK1-deficient mice exhibited an elevated arterial blood pressure, which was most prominent during physical activity. Overexpression of SK3 in IK1−/−/SK3T/T mice partially restored EDHF- and nitric oxide–mediated vasodilation and lowered elevated blood pressure. The IK1-opener SKA-31 enhanced EDHF-mediated vasodilation and lowered blood pressure in SK3-deficient IK1+/+/SK3T/T (+doxycycline) mice to normotensive levels. Conclusions— Our study demonstrates that endothelial SK3 and IK1 channels have distinct stimulus-dependent functions, are major players in the EDHF pathway, and significantly contribute to arterial blood pressure regulation. Endothelial KCa channels may represent novel therapeutic targets for the treatment of hypertension.

Selective blockade of endothelial Ca2+‐activated small‐ and intermediate‐conductance K+‐channels suppresses EDHF‐mediated vasodilation

Activation of Ca2+‐activated K+‐channels (KCa) has been suggested to play a key role in endothelium‐derived hyperpolarizing factor (EDHF)‐mediated vasodilation. However, due to the low selectivity of commonly used KCa‐channel blockers it is still elusive which endothelial KCa‐subtypes mediate hyperpolarization and thus initiate EDHF‐mediated vasodilation. Using the non‐cytochrome P450 blocking clotrimazole‐derivatives, 1‐[(2‐chlorophenyl) diphenylmethyl]‐1H‐pyrazole (TRAM‐34) and 2‐(2‐chlorophenyl)‐2,2‐diphenylacetonitrile (TRAM‐39) as highly selective IK1‐inhibitors, we investigated the role of the intermediate‐conductance KCa (rIK1) in endothelial hyperpolarization and EDHF‐mediated vasodilation. Expression and function of rIK1 and small‐conductance KCa (rSK3) were demonstrated in situ in single endothelial cells of rat carotid arteries (CA). rIK1‐currents were blocked by TRAM‐34 or TRAM‐39, while rSK3 was blocked by apamin. In current‐clamp experiments, endothelial hyperpolarization in response to acetylcholine was abolished by the combination of apamin and TRAM‐34. In phenylephrine‐preconstricted CA, acetylcholine‐induced NO and prostacyclin‐independent vasodilation was almost completely blocked by ChTX, CLT, TRAM‐34, or TRAM‐39 in combination with the SK3‐blocker apamin. Apamin, TRAM‐34, and CLT alone or sulphaphenzole, a blocker of the cytochrome P450 isoform 2C9, were ineffective in blocking the EDHF‐response. In experiments without blocking NO and prostacyclin synthesis, the combined blockade of SK3 and IK1 reduced endothelium‐dependent vasodilation. In conclusion, the use of selective IK1‐inhibitors together with the SK3‐blocker apamin revealed that activation of both KCa, rIK1 and rSK3 is crucial in mediating endothelial hyperpolarization and generation of the EDHF‐signal while the cytochrome P450 pathway seems to play a minor or no role in rat CA.

Endothelial Ca2+-activated K+ channels in normal and impaired EDHF–dilator responses – relevance to cardiovascular pathologies and drug discovery

The arterial endothelium critically contributes to blood pressure control by releasing vasodilating autacoids such as nitric oxide, prostacyclin and a third factor or pathway termed ‘endothelium‐derived hyperpolarizing factor’ (EDHF). The nature of EDHF and EDHF‐signalling pathways is not fully understood yet. However, endothelial hyperpolarization mediated by the Ca2+‐activated K+ channels (KCa) has been suggested to play a critical role in initializing EDHF–dilator responses in conduit and resistance‐sized arteries of many species including humans. Endothelial KCa currents are mediated by the two KCa subtypes, intermediate‐conductance KCa (KCa3.1) (also known as, a.k.a. IKCa) and small‐conductance KCa type 3 (KCa2.3) (a.k.a. SKCa). In this review, we summarize current knowledge about endothelial KCa3.1 and KCa2.3 channels, their molecular and pharmacological properties and their specific roles in endothelial function and, particularly, in the EDHF–dilator response. In addition we focus on recent experimental evidences derived from KCa3.1‐ and/or KCa2.3‐deficient mice that exhibit severe defects in EDHF signalling and elevated blood pressures, thus highlighting the importance of the KCa3.1/KCa2.3‐EDHF–dilator system for blood pressure control. Moreover, we outline differential and overlapping roles of KCa3.1 and KCa2.3 for EDHF signalling as well as for nitric oxide synthesis and discuss recent evidence for a heterogeneous (sub) cellular distribution of KCa3.1 (at endothelial projections towards the smooth muscle) and KCa2.3 (at inter‐endothelial borders and caveolae), which may explain their distinct roles for endothelial function. Finally, we summarize the interrelations of altered KCa3.1/KCa2.3 and EDHF system impairments with cardiovascular disease states such as hypertension, diabetes, dyslipidemia and atherosclerosis and discuss the therapeutic potential of KCa3.1/KCa2.3 openers as novel types of blood pressure‐lowering drugs.

Selective Blockade of the Intermediate-Conductance Ca2+-Activated K+ Channel Suppresses Proliferation of Microvascular and Macrovascular Endothelial Cells and Angiogenesis In Vivo

Objective—Ca2+-activated K+ (KCa) channels have been proposed to promote mitogenesis in several cell types. Here, we tested whether the intermediate-conductance KCa channel (IKCa1) and the large-conductance KCa channel (BKCa) contribute to endothelial cell (EC) proliferation and angiogenesis. Material and Results—Function and expression of IKCa1 and BKCa/Slo were investigated by patch-clamp analysis and real-time RT-PCR in human umbilical vein ECs (HUVECs) and in dermal human microvascular ECs 1 (HMEC-1). HMEC-1 expressed IKCa1 and BKCa/Slo, whereas HUVECs expressed IKCa1. A 48-hour exposure to basic fibroblast growth factor (bFGF) augmented IKCa1 current amplitudes and induced a 3-fold increase in IKCa1 mRNA expression in HUVECs and HMEC-1. Vascular endothelial growth factor (VEGF) was also effective in upregulating IKCa1. BKCa/Slo expression and current amplitudes in HMEC-1 were not altered by bFGF. bFGF- and VEGF-induced EC proliferation was suppressed by charybdotoxin, clotrimazole, or the selective IKCa1 blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), whereas inhibition of BKCa/Slo by iberiotoxin was ineffective. In the Matrigel plug assay in mice, administration of TRAM-34 for 2 weeks significantly suppressed angiogenesis by ≈85%. Conclusions—bFGF and VEGF upregulate expression of IKCa1 in human ECs. This upregulation of IKCa1 seems to be required for mitogen-induced EC proliferation and angiogenesis in vivo. Selective IKCa1 blocker might be of therapeutic value to prevent tumor angiogenesis.

Chronic epithelial kidney injury molecule-1 expression causes murine kidney fibrosis

Acute kidney injury predisposes patients to the development of both chronic kidney disease and end-stage renal failure, but the molecular details underlying this important clinical association remain obscure. We report that kidney injury molecule-1 (KIM-1), an epithelial phosphatidylserine receptor expressed transiently after acute injury and chronically in fibrotic renal disease, promotes kidney fibrosis. Conditional expression of KIM-1 in renal epithelial cells (Kim1(RECtg)) in the absence of an injury stimulus resulted in focal epithelial vacuolization at birth, but otherwise normal tubule histology and kidney function. By 4 weeks of age, Kim1(RECtg) mice developed spontaneous and progressive interstitial kidney inflammation with fibrosis, leading to renal failure with anemia, proteinuria, hyperphosphatemia, hypertension, cardiac hypertrophy, and death, analogous to progressive kidney disease in humans. Kim1(RECtg) kidneys had elevated expression of proinflammatory monocyte chemotactic protein-1 (MCP-1) at early time points. Heterologous expression of KIM-1 in an immortalized proximal tubule cell line triggered MCP-1 secretion and increased MCP-1-dependent macrophage chemotaxis. In mice expressing a mutant, truncated KIM-1 polypeptide, experimental kidney fibrosis was ameliorated with reduced levels of MCP-1, consistent with a profibrotic role for native KIM-1. Thus, sustained KIM-1 expression promotes kidney fibrosis and provides a link between acute and recurrent injury with progressive chronic kidney disease.

Renal fibrosis is attenuated by targeted disruption of KCa3.1 potassium channels

Proliferation of interstitial fibroblasts is a hallmark of progressive renal fibrosis commonly resulting in chronic kidney failure. The intermediate-conductance Ca2+-activated K+ channel (KCa3.1) has been proposed to promote mitogenesis in several cell types and contribute to disease states characterized by excessive proliferation. Here, we hypothesized that KCa3.1 activity is pivotal for renal fibroblast proliferation and that deficiency or pharmacological blockade of KCa3.1 suppresses development of renal fibrosis. We found that mitogenic stimulation up-regulated KCa3.1 in murine renal fibroblasts via a MEK-dependent mechanism and that selective blockade of KCa3.1 functions potently inhibited fibroblast proliferation by G0/G1 arrest. Renal fibrosis induced by unilateral ureteral obstruction (UUO) in mice was paralleled by a robust up-regulation of KCa3.1 in affected kidneys. Mice lacking KCa3.1 (KCa3.1−/−) showed a significant reduction in fibrotic marker expression, chronic tubulointerstitial damage, collagen deposition and αSMA+ cells in kidneys after UUO, whereas functional renal parenchyma was better preserved. Pharmacological treatment with the selective KCa3.1 blocker TRAM-34 similarly attenuated progression of UUO-induced renal fibrosis in wild-type mice and rats. In conclusion, our data demonstrate that KCa3.1 is involved in renal fibroblast proliferation and fibrogenesis and suggest that KCa3.1 may represent a therapeutic target for the treatment of fibrotic kidney disease.