Kathryn Smedlund

The Constitutive Function of Native TRPC3 Channels Modulates Vascular Cell Adhesion Molecule-1 Expression in Coronary Endothelial Cells Through Nuclear Factor κB Signaling

Rationale: Upregulation of endothelial vascular cell adhesion molecule (VCAM)-1 and the subsequent increase in monocyte recruitment constitute critical events in atherogenesis. We have recently shown that in human coronary artery endothelial cells (HCAECs) regulated expression of VCAM-1 depends, to a significant extent, on expression and function of the Ca2+-permeable channel transient receptor potential canonical (TRPC)3, regardless of the ability of the stimulatory signal to induce regulated Ca2+ influx, leading to the hypothesis that TRPC3 constitutive, rather than regulated function, contributes to the underlying signaling mechanism. Objective: The present studies addressed this important question and gathered mechanistic insight on the signaling coupling constitutive TRPC3 function to VCAM-1 expression. Methods and Results: In HCAECs, maneuvers that prevent Ca2+ influx or knockdown of TRPC3 markedly reduced tumor necrosis factor (TNF)&agr;-induced VCAM-1 and monocyte adhesion. TNF&agr; also induced TRPC3 expression and TRPC3-mediated constitutive cation influx and currents. Stable (HEK293 cells) or transient (HCAECs) overexpression of TRPC3 enhanced TNF&agr;-induced VCAM-1 compared to wild-type cells. I&kgr;B&agr; phosphorylation/degradation was reduced by TRPC3 knockdown and increased by channel overexpression. Inhibition of calmodulin completely prevented nuclear factor &kgr;B activation, whereas blocking calmodulin-dependent kinases or NADPH oxidases rendered partial inhibition. Conclusions: Our findings indicate that in HCAECs expression of VCAM-1 and monocyte adhesion depend, to a significant extent, on TRPC3 constitutive function through a signaling mechanism that requires constitutive TRPC3-mediated Ca2+ influx for proper activation of nuclear factor &kgr;B, presumably through Ca2+-dependent activation of the calmodulin/calmodulin-dependent kinase axis.

Involvement of Native TRPC3 Proteins in ATP-Dependent Expression of VCAM-1 and Monocyte Adherence in Coronary Artery Endothelial Cells

Background—Vascular cell adhesion molecule-1 (VCAM-1) is critical in monocyte recruitment to the endothelium, a key event in development of atherosclerotic lesions. Stimulation of human coronary artery endothelial cells (HCAECs) with ATP positively modulates VCAM-1 expression and function through a mechanism involving Ca2+ signaling. We here examined the role of Ca2+ influx and native TRPC3 channels in that mechanism. Methods and Results—Omission of extracellular Ca2+ or pretreatment of cells with channel blockers markedly reduced ATP-induced VCAM-1 and monocyte adhesion. Using a siRNA strategy and real-time fluorescence, we found that native TRPC3 proteins contribute to constitutive and ATP-regulated Ca2+ influx. ATP-dependent upregulation of VCAM-1 was accompanied by an increase in basal cation entry and TRPC3 expression. Notably, TRPC3 knock-down resulted in a dramatic reduction of ATP-induced VCAM-1 and monocyte adhesion. Conclusions—These findings indicate that in HCAECs, native TRPC3 proteins form channels that contribute to constitutive and ATP-dependent Ca2+ influx, and that TRPC3 expression and function are fundamental to support VCAM-1 expression and monocyte binding. This is the first evidence to date relating native TRPC3 proteins with regulated expression of cell adhesion molecules in coronary endothelium, and suggests a potential pathophysiological role of TRPC3 in coronary artery disease.

Impairment of survival signaling and efferocytosis in TRPC3-deficient macrophages.

We have recently shown that in macrophages proper operation of the survival pathways phosphatidylinositol-3-kinase (PI3K)/AKT and nuclear factor kappa B (NFkB) has an obligatory requirement for constitutive, non-regulated Ca(2+) influx. In the present work we examined if Transient Receptor Potential Canonical 3 (TRPC3), a member of the TRPC family of Ca(2+)-permeable cation channels, contributes to the constitutive Ca(2+) influx that supports macrophage survival. We used bone marrow-derived macrophages obtained from TRPC3(-/-) mice to determine the activation status of survival signaling pathways, apoptosis and their efferocytic properties. Treatment of TRPC3(+/+) macrophages with the pro-apoptotic cytokine TNFα induced time-dependent phosphorylation of IκBα, AKT and BAD, and this was drastically reduced in TRPC3(-/-) macrophages. Compared to TRPC3(+/+) cells TRPC3(-/-) macrophages exhibited reduced constitutive cation influx, increased apoptosis and impaired efferocytosis. The present findings suggest that macrophage TRPC3, presumably through its constitutive function, contributes to survival signaling and efferocytic properties.

Bone marrow deficiency of TRPC3 channel reduces early lesion burden and necrotic core of advanced plaques in a mouse model of atherosclerosis.

AIMS Macrophage apoptosis plays a determinant role in progression of atherosclerotic lesions. An important goal in atherosclerosis research is to identify new components of macrophage apoptosis that can eventually be exploited as molecular targets in strategies aimed at manipulating macrophage function in the lesion. In the previous work from our laboratory, we have shown that transient receptor potential canonical 3 (TRPC3) channel is an obligatory component of survival mechanisms in human and murine macrophages and that TRPC3-deficient non-polarized bone marrow-derived macrophages exhibit increased apoptosis, suggesting that in vivo TRPC3 might influence lesion development. In the present work, we used a bone marrow transplantation strategy as a first approach to examine the impact of macrophage deficiency of TRPC3 on early and advanced atherosclerotic lesions of Apoe(-/-) mice. METHODS AND RESULTS After 3 weeks of high-fat diet, lesions in mice transplanted with bone marrow from Trpc3(-/-) donors were smaller and with reduced cellularity than controls. Advanced lesions from these mice exhibited reduced necrotic core, less apoptotic macrophages, and increased collagen content and cap thickness. In vitro, TRPC3-deficient macrophages polarized to the M1 phenotype showed reduced apoptosis, whereas both M1 and M2 macrophages had increased efferocytic capacity. CONCLUSIONS Bone marrow deficiency of TRPC3 has a dual beneficial effect on lesion progression by reducing cellularity at early stages and necrosis in the advanced plaques. Our findings represent the first evidence for a role of a member of the TRPC family of cation channels in mechanisms associated with atherosclerosis.

On the potential role of source and species of diacylglycerol in phospholipase-dependent regulation of TRPC3 channels

Members of the Transient Receptor Potential Canonical (TRPC) family of channel forming proteins are among the most important Ca2+-permeable cation channels in non-excitable cells. Physiologically, TRPC channels are activated downstream receptor-dependent stimulation of phospholipases, either by store-operated or non-store operated mechanisms. TRPC3, a member of the TRPC3/6/7 subfamily, has been largely studied mostly due to its ability to function in one or the other modes, depending on cell type and expression conditions. The role of TRPC3 as a non-store operated channel has been attributed to its ability to respond to diacylglycerol (DAG) either exogenously applied or endogenously produced following activation of receptor-stimulated phospholipases. Despite the vast amount of information accumulated on this topic, some critical aspects related to phospholipase-dependent DAG-mediated regulation of TRPC3 remain unclear and/or unexplored. Among these, the source and species of native DAG, modulation by different DAG-generating phospholipases and protein kinase C-dependent inhibition of TRPC3 in its native environment are just few examples. The present essay is intended to compile existing knowledge on the nature of phospholipase-derived DAGs, their biophysical properties and current evidence on phospholipase-dependent regulation of TRPC3, to speculate on potential scenarios that may eventually provide answers to some of the above questions.

Increased size and cellularity of advanced atherosclerotic lesions in mice with endothelial overexpression of the human TRPC3 channel

Significance Atherosclerosis is a chronic disease of the arterial wall with a dominant inflammatory component. Endothelial cell inflammation and recruitment of circulating monocytes are critical processes during atherosclerotic lesion progression. In this manuscript, we generated a mouse model of atherosclerosis with endothelial-specific overexpression of TRPC3, a calcium permeable channel, and provide evidence indicating that augmented expression/function of TRPC3 supports, in vivo, endothelial inflammation and increased macrophage infiltration, resulting in atherosclerotic lesions of bigger size and complexity. These findings support the notion that endothelial TRPC3 channels may represent attractive targets for development of novel therapeutic strategies in the treatment of atherosclerosis. In previous in vitro studies, we showed that Transient Receptor Potential Canonical 3 (TRPC3), a calcium-permeable, nonselective cation channel endowed with high constitutive function, is an obligatory component of the inflammatory signaling that controls expression of the vascular cell adhesion molecule-1 (VCAM-1) and monocyte adhesion to coronary artery endothelial cells. Also, TRPC3 expression in these cells was found to be up-regulated by proatherogenic factors, which enhanced inflammation and VCAM-1 expression. However, it remained to be determined whether these in vitro findings were of relevance to atherosclerotic lesion development in vivo. To answer this important question in the present work, we generated mice with endothelial-specific overexpression of human TRPC3 in an Apoe knockout background (TgEST3ApoeKO) and examined lesions in the aortic sinus following 10 and 16 wk on a high-fat diet. No significant differences were found in size or complexity of early stage lesions (10 wk). However, advanced plaques (16 wk) from TgEST3ApoeKO mice exhibited a significant increase in size and macrophage content compared with nontransgenic littermate controls. Remarkably, this change was correlated with increased VCAM-1 and phospho-IkBα immunoreactivity along the endothelial lining of lesions from transgenic animals compared with controls. These findings validate the in vivo relevance of previous in vitro findings and represent, to our knowledge, the first in vivo evidence for a proatherogenic role of endothelial TRPC3.

Evidence for Operation of Nicotinic and Muscarinic Acetylcholine Receptor-Dependent Survival Pathways in Human Coronary Artery Endothelial Cells

Nicotinic acetylcholine receptors (nAChRs) have recently emerged as critical players in modulation of endothelial function. In particular, studies on endothelial cells from different vascular beds have shown anti‐apoptotic actions of nicotinic stimulation, but whether there is actually activation of survival signaling downstream nAChR function has not been explored. In the present work we used human coronary artery endothelial cells (HCAECs) and a pharmacological approach to examine the impact of cholinergic stimulation on survival signaling pathways. Our findings show that cholinergic receptors promote activation of three typical survival routes: the phosphatidyl‐inositol‐3‐kinase (PI3K)/AKT axis, activated downstream muscarinic and nAChRs; the JAK2/STAT3 axis, activated downstream nAChR; and ERK1/2 MAP kinases, activated by both muscarinic acetylcholine receptor (mAChR) and nAChR. Based on their sensitivity to α‐bungarotoxin, nicotinic regulation of JAK2/STAT3 and ERK1/2 occurs downstream α7‐nAChRs. The present findings suggest that in HCAECs the two cholinergic receptors may act concertedly to induce an efficient survival response of coronary cells when exposed to pro‐apoptotic stimuli. J. Cell. Biochem. 112: 1978–1984, 2011.

Pharmacological evidence for a role of the transient receptor potential canonical 3 (TRPC3) channel in endoplasmic reticulum stress-induced apoptosis of human coronary artery endothelial cells

Unresolved endoplasmic reticulum (ER) stress, with the subsequent persistent activation of the unfolded protein response (UPR) is a well-recognized mechanism of endothelial cell apoptosis with a major impact on the integrity of the endothelium during the course of cardiovascular diseases. As in other cell types, Ca(2+) influx into endothelial cells can promote ER stress and/or contribute to mechanisms associated with it. In previous work we showed that in human coronary artery endothelial cells (HCAECs) the Ca(2+)-permeable non-selective cation channel Transient Receptor Potential Canonical 3 (TRPC3) mediates constitutive Ca(2+) influx which is critical for operation of inflammatory signaling in these cells, through a mechanism that entails coupling of TRPC3 constitutive function to activation of Ca(2+)/calmodulin-dependent protein kinase II (CAMKII). TRPC3 has been linked to UPR signaling and apoptosis in cells other than endothelial, and CAMKII is a mediator of ER stress-induced apoptosis in various cell types, including endothelial cells. In the present work we used a pharmacological approach to examine whether in HCAECs TRPC3 and CAMKII also contribute to mechanisms of ER stress-induced apoptosis. The findings show for the first time that in HCAECs activation of the UPR and the subsequent ER stress-induced apoptosis exhibit a strong requirement for constitutive Ca(2+) influx and that TRPC3 contributes to this process. In addition, we obtained evidence indicating that, similar to its roles in non-endothelial cells, CAMKII participates in ER stress-induced apoptosis in HCAECs.

Pleiotropic Effect of a High Resolution Mapped Blood Pressure QTL on Tumorigenesis.

This study is focused on a translationally significant, genome-wide-association-study (GWAS) locus for cardiovascular disease (QT-interval) on human chromosome 17. We have previously validated and high resolution mapped the homologous genomic segment of this human locus to <42.5 kb on rat chromosome 10. This <42.5 kb segment in rats regulates both QT-interval and blood pressure and contains a single protein-coding gene, rififylin (Rffl). The expression of Rffl in the hearts and kidneys is differential between Dahl S and S.LEW congenic rats, which are the strains used for mapping this locus. Our previous study points to altered rate of endocytic recycling as the underlying mechanism, through which Rffl operates to control both QT-interval and blood pressure. Interestingly, Rffl also contributes to tumorigenesis by repressing caspases and tumor suppressor genes. Moreover, the expression of Methyl-CpG Binding Domain Protein 2 (Mbd2) in the hearts and kidneys is also higher in the S.LEW congenic strain than the background (control) Dahl S strain. Mbd2 can repress methylated tumor suppressor genes. These data suggest that the S.LEW congenic strain could be more susceptible to tumorigenesis. To test this hypothesis, the S and S.LEW strains were compared for susceptibility to azoxymethane-induced colon tumors. The number of colon tumors was significantly higher in the S.LEW congenic strain compared with the S rat. Transcriptomic analysis confirmed that the chemical carcinogenesis pathway was significantly up-regulated in the congenic strain. These studies provide evidence for a GWAS-validated genomic segment on rat chromosome 10 as being important for the regulation of cardiovascular function and tumorigenesis.

Molecular and Cellular Aspects of Atherosclerosis: Emerging Roles of TRPC Channels

Recruitment of circulating monocytes to activated areas of the endothelium and their migration to the subintimal inflammatory foci represents one of the earliest events in atherogenesis (Linton and Fazio 2003; Hansson 2005). Importantly, monocyte recruitment can be recognized throughout all lesional stages including advanced lesions, where plaque infiltration and neovascularization occur. Indeed, available experimental evidence supports the notion that in advanced stages monocyte infiltration contributes to plaque instability and rupture (Virmani, Burke et al. 2006). Monocyte recruitment to the subendothelial milieu implies a sequence of events that begin with monocyte rolling along and tethering to the endothelial surface, firm adhesion and activation, and ultimately migration to the subintima. At the molecular level, the entire sequence entails interaction of integrins on the monocyte surface with cell adhesion molecules (CAMs) expressed on the endothelial cell. Monocyte rolling and tethering is mainly mediated by CAMs from the selectin group (v.g., E-selectin) while firm adhesion and migration are mostly mediated by CAMs from the immunoglobulin (Ig) superfamily, such as intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Compelling evidence accumulated over the last decade has clearly established that VCAM-1 (CD106) has a prominent role in mediating attachment and migration of monocytes (the Cluster of Differentiation nomenclature (Zola, Swart et al. 2005) is given for reference, but “VCAM-1” will be used throughout the text).