Brajesh P. Kaistha

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.

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.

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.

Vascular KCa-channels as therapeutic targets in hypertension and restenosis disease

Importance of the field: Cardiovascular disease is a leading cause of death in modern societies. Hyperpolarizing Ca2+-activated K+ channels (KCa) are important membrane proteins in the control of arterial tone and pathological vascular remodelling and thus could serve as new drug targets. Areas covered in this review: We summarize recent advances in the field of vascular KCa and their roles in cardiovascular pathologies such as hypertension and restenosis disease and draw attention to novel small-molecule channel modulators and their possible therapeutic utility. This review focuses on literature from the last four to five years. What the reader will gain: Pharmacological opening of endothelial KCa3.1/KCa2.3 channels stimulates endothelium-derived-hyperpolarizing-factor-mediated arteriolar dilation and lowers blood pressure. Inhibition of smooth muscle KCa3.1 channels has beneficial effects in restenosis disease and atherosclerosis. We consider the therapeutic potential of KCa3.1/KCa2.3 openers as novel endothelium-specific antihypertensive drugs as well as of KCa3.1-blockers for the treatment of pathological vascular remodelling and discuss advantages and disadvantages of the pharmacotherapeutic approaches. Take home message: Pharmacological manipulation of vascular KCa channels by novel small-molecule modulators offers new venues for alternative treatments of hypertension, restenosis and atherosclerosis. Additional efforts are required to optimize these compounds and to validate them as cardiovascular-protective drugs.

PLAC8 Localizes to the Inner Plasma Membrane of Pancreatic Cancer Cells and Regulates Cell Growth and Disease Progression through Critical Cell-Cycle Regulatory Pathways

Pancreatic ductal adenocarcinoma (PDAC) carries the most dismal prognosis of all solid tumors and is generally strongly resistant to currently available chemo- and/or radiotherapy regimens, including targeted molecular therapies. Therefore, unraveling the molecular mechanisms underlying the aggressive behavior of pancreatic cancer is a necessary prerequisite for the development of novel therapeutic approaches. We previously identified the protein placenta-specific 8 (PLAC8, onzin) in a genome-wide search for target genes associated with pancreatic tumor progression and demonstrated that PLAC8 is strongly ectopically expressed in advanced preneoplastic lesions and invasive human PDAC. However, the molecular function of PLAC8 remained unclear, and accumulating evidence suggested its role is highly dependent on cellular and physiologic context. Here, we demonstrate that in contrast to other cellular systems, PLAC8 protein localizes to the inner face of the plasma membrane in pancreatic cancer cells, where it interacts with specific membranous structures in a temporally and spatially stable manner. Inhibition of PLAC8 expression strongly inhibited pancreatic cancer cell growth by attenuating cell-cycle progression, which was associated with transcriptional and/or posttranslational modification of the central cell-cycle regulators CDKN1A, retinoblastoma protein, and cyclin D1 (CCND1), but did not impact autophagy. Moreover, Plac8 deficiency significantly inhibited tumor formation in genetically engineered mouse models of pancreatic cancer. Together, our findings establish PLAC8 as a central mediator of tumor progression in PDAC and as a promising candidate gene for diagnostic and therapeutic targeting.

The deubiquitinating enzyme USP5 promotes pancreatic cancer via modulating cell cycle regulators

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid tumors. With an overall five-year survival rate remaining below 6%, there is an explicit need to search for new molecular targets for therapeutic interventions. We undertook a barcode labelled short-hairpin (shRNA) library screen in pancreatic cancer cells in order to identify novel genes promoting cancer survival and progression. Among the candidate genes identified in this screen was the deubiquitinase USP5, which subsequent gene expression analyses demonstrated to be significantly upregulated in primary human pancreatic cancer tissues. Using different knockdown approaches, we show that expression of USP5 is essential for the proliferation and survival of pancreatic cancer cells, tested under different 2D and 3D cell culture conditions as well as in in vivo experiments. These growth inhibition effects upon knockdown of USP5 are mediated primarily by the attenuation of G1/S phase transition in the cells, which is accompanied by accumulation of DNA damage, upregulation of p27, and increased apoptosis rates. Since USP5 is overexpressed in cancer tissues, it can thus potentially serve as a new target for therapeutic interventions, especially given the fact that deubiquitinases are currently emerging as new class of attractive drug targets in cancer.