Selective Blockade of the Intermediate-Conductance Ca ²⁺ -Activated K ⁺ Channel Suppresses Proliferation of Microvascular and Macrovascular Endothelial Cells and Angiogenesis In Vivo

Abstract

Objective-Ca2ϩ -activated K ϩ (K Ca ) channels have been proposed to promote mitogenesis in several cell types. Here, we tested whether the intermediate-conductance K Ca channel (IKCa1) and the large-conductance K Ca channel (BK Ca ) contribute to endothelial cell (EC) proliferation and angiogenesis. Material and Results-Function and expression of IKCa1 and BK Ca /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 ngiogenesis is an important process in a variety of physiological and pathophysiological conditions such as embryonic vasculogenesis, wound healing, poststenotic collateral formation, inflammation, and tumor vascularization. 1 Regarding the latter, tumor-derived angiogenic factors such as the vascular endothelial growth factor (VEGF) and the basic fibroblast growth factor (bFGF) induce endothelial cell (EC) sprouting, proliferation, migration, and finally new vessel formation. 1,2 With respect to EC proliferation, several studies have shown that EC proliferation after stimulation with angiogenic factors is initiated by a rise in intracellular calcium mediated through calcium-influx channels. [3][4][5] By keeping the membrane potential negative, chloride and potassium channels provide the driving force for this calcium entry and thus play an important role in regulating cell cycle progression 6 -9 as well as angiogenesis, as shown previously for chloride channels. 6 In particular, the intermediate- Materials and Methods Human ECs and In Vitro Proliferation StudiesHUVECs were isolated as described previously. 23 HUVECs and HMEC-1, a cell line derived from dermal HMECs 24 were cultured as described previously. 23 For proliferation and expression studies, HUVECs of second passage were used to avoid senescence.To induce growth arrest, HUVECs were kept in low serum medium (2% FCS) to allow cell survival for 48 hours before stimulation with bFGF (50 ng/mL) or VEGF-165 (50 ng/mL). HMEC-1 were kept in serum-free medium for 48 hours before stimulation with bFGF (50 ng/mL). At 5% to 10% confluence, photomicrographs of cells were taken in a fixed field before and 48 hours after stimulation and the percentage increase in cell count was calculated for each experiment. For blocker studies, cells were treated with TRAM-34 (0.1 nmol/L to 1 mol/L), 12 the inactive analogue TRAM-7 (1-tritylpyrrolidine; 1 mol/L), 12 CLT (0.1 nmol/L to 1 mol/L), or iberiotoxin (IbTx; 100 nmol/L). ReagentsPD98059, SB203580, and SP600125 were obtained from Tocris. CLT, IbTx, charydotoxin (ChTx), and apamin were obtained from Sigma. bFGF and VEGF-165 were obtained from Biochrom. All other chemicals were obtained from Sigma.

SK4 channels modulate Ca2+ signalling and cell cycle progression in murine breast cancer

“…The SK4‐dependent current fraction (difference between red and black symbols in Fig. 1G) exhibited strong inward rectification as reported for canonical SK4 channels which are usually present at the plasma membrane of, for example, endothelial cells and smooth muscle cells (Grgic et al ., 2005; Toyama et al ., 2008). …”

“…Current knowledge suggests that BK (Lallet‐Daher et al ., 2009; Parihar et al ., 2003; Stegen et al ., 2015) and SK4 (Ouadid‐Ahidouch et al ., 2004; Parihar et al ., 2003; Steinle et al ., 2011) activities are required for malignant growth of several tumour‐derived cell lines and of xenografts in immunocompromised mice, highlighting a general role of K Ca channels for cell cycle‐specific functions (Huang and Jan, 2014; Pardo and Stuhmer, 2014). Expression of SK4 and BK in cancer cells follows a cell cycle‐dependent mode (Ouadid‐Ahidouch et al ., 2004; Pardo et al ., 1998) and the mitogen‐dependent regulation of K Ca activity supports a role for both channels in malignant (Faouzi et al ., 2010; Lallet‐Daher et al ., 2009; Wang et al ., 2007a) and nonmalignant cell proliferation (Grgic et al ., 2005; Khanna et al ., 1999; Toyama et al ., 2008; Yu et al ., 2013). By inducing a more negative membrane voltage, activation of K + channels provides a driving force for Ca 2+ influx into the nonexcitable tumour cell.…”

Renal fibrosis is attenuated by targeted disruption of K Ca 3.1 potassium channels

“…Conversely, we and others have recently demonstrated in models of experimental angiogenesis (15), post-interventional arterial restenosis (17,19), atherosclerosis (26), and endometrial cancer (27), that selective pharmacological inhibition or knockdown of K Ca 3.1 suppresses mitogen-driven cell proliferation and ameliorates disease progression. Thus, K Ca 3.1 could have a pivotal role in disease states characterized by excessive cell proliferation and may emerge as a promising pharmacotherapeutic target for antiproliferative treatment.…”

“…Interestingly, mitogens such as bFGF, PDGF, or VEGF have been found to distinctly up-regulate K Ca 3.1 in several cell types (15,21). Conversely, we and others have recently demonstrated in models of experimental angiogenesis (15), post-interventional arterial restenosis (17,19), atherosclerosis (26), and endometrial cancer (27), that selective pharmacological inhibition or knockdown of K Ca 3.1 suppresses mitogen-driven cell proliferation and ameliorates disease progression.…”

“…Whereas the first directly mediate Ca 2ϩ inflow, the latter regulate membrane potential and thus provide the driving force for Ca 2ϩ entry (13). In particular, the intermediate-conductance K Ca (K Ca 3.1) has been shown to play an important role in promoting mitogenesis in several tissues such as the endothelium (15,16), vascular smooth muscle (17)(18)(19), and T lymphocytes (20) as well as in several cell lines including A7r5 neonatal aortic smooth muscle cells (21), 10T1/2-MRF4 myogenic fibroblasts (22,23), HMEC-1 dermal endothelial cells (15), and some cancer cell lines (24,25).…”