Guillermo Vazquez

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.

Requirement for non-regulated, constitutive calcium influx in macrophage survival signaling

The phosphatidylinositol-3-kinase (PI3K)/AKT axis and the Nuclear Factor kappa B (NFκB) pathway play critical roles in macrophage survival. In cells other than macrophages proper operation of those two pathways requires Ca²(+) influx into the cell, but if that is the case in macrophages remains unexplored. In the present work we used THP-1-derived macrophages and a pharmacological approach to examine for the first time the role of constitutive, non-regulated Ca²(+) influx in PI3K/AKT and NFκB signaling. Blocking constitutive function of Ca²(+)-permeable channels with the organic channel blocker SKF96365 completely prevented phosphorylation of IκBα, AKT and its downstream target BAD in TNFα-treated macrophages. A similar effect was observed upon treating macrophages with the calmodulin (CAM) inhibitor W-7 or the calmodulin-dependent kinase II (CAMKII) inhibitor KN-62. In addition, pre-treating macrophages with SKF96365 significantly enhanced TNFα-induced apoptosis. Our findings suggest that in THP-1-derived macrophages survival signaling depends, to a significant extent, on constitutive Ca²(+) influx presumably through a mechanism that involves the CAM/CAMKII axis as a coupling component between constitutive Ca²(+) influx and activation of survival signaling.

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.

Macrophage function in atherosclerosis: potential roles of TRP channels.

Cation channels of the Transient Receptor Potential Canonical (TRPC) group, which belong to the larger TRP superfamily of channel proteins, are critical players in cardiovascular disease. Recent studies underscored a role of TRPC3 in macrophage survival and efferocytosis, two critical events in atherosclerosis lesion development. Also, other members of the TRP channel superfamily are found expressed in monocytes/macrophages, where they participate in processes that might be of significance to atherogenesis. These observations set a framework for future studies aimed at defining the ultimate functions not only of TRPC3, but probably other TRP channels, in macrophage biology. The purpose of this manuscript is to provide a timely revision of existing evidence on the role of members of the TRP channel superfamily, in particular TRPCs, in macrophages and discuss it in the context of the macrophage’s function in atherogenesis.

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.

Ceacam1 deletion causes vascular alterations in large vessels

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes hepatic insulin clearance and endothelial survival. However, its role in the morphology of macrovessels remains unknown. Mice lacking Ceacam1 (Cc1-/-) exhibit hyperinsulinemia, which causes insulin resistance and fatty liver. With increasing evidence of an association among hyperinsulinemia, fatty liver disease, and atherosclerosis, we investigated whether Cc1-/- exhibited vascular lesions in atherogenic-prone aortae. Histological analysis revealed impaired endothelial integrity with restricted fat deposition and aortic plaque-like lesions in Cc1-/- aortae, likely owing to their limited lipidemia. Immunohistochemical analysis indicated macrophage deposition, and in vitro studies showed increased leukocyte adhesion to aortic wall, mediated in part by elevation in vascular cell adhesion molecule 1 levels. Basal aortic eNOS protein and NO content were reduced, in parallel with reduced Akt/eNOS and Akt/Foxo1 phosphorylation. Ligand-induced vasorelaxation was compromised in aortic rings. Increased NADPH oxidase activity and plasma 8-isoprostane levels revealed oxidative stress and lipid peroxidation in Cc1-/- aortae. siRNA-mediated CEACAM1 knockdown in bovine aortic endothelial cells adversely affected insulins stimulation of IRS-1/PI 3-kinase/Akt/eNOS activation by increasing IRS-1 binding to SHP2 phosphatase. This demonstrates that CEACAM1 regulates both endothelial cell autonomous and nonautonomous mechanisms involved in vascular morphology and NO production in aortae. Systemic factors such as hyperinsulinemia could contribute to the pathogenesis of these vascular abnormalities. Cc1-/- mice provide a first in vivo demonstration of distinct CEACAM1-dependent hepatic insulin clearance linking hepatic to macrovascular abnormalities.

Reduced endoplasmic reticulum stress-induced apoptosis and impaired unfolded protein response in TRPC3-deficient M1 macrophages

Endoplasmic reticulum (ER) stress is a prominent mechanism of macrophage apoptosis in advanced atherosclerotic lesions. Recent studies from our laboratory showed that advanced atherosclerotic plaques in Apoe(-/-) mice with bone marrow deficiency of the calcium-permeable channel Transient Receptor Potential Canonical 3 (TRPC3) are characterized by reduced areas of necrosis and fewer apoptotic macrophages than animals transplanted with Trpc3(+/+) bone marrow. In vitro, proinflammatory M1 but not anti-inflammatory M2 macrophages derived from Trpc3(-/-)Apoe(-/-) animals exhibited reduced ER stress-induced apoptosis. However, whether this was due to a specific effect of TRPC3 deficiency on macrophage ER stress signaling remained to be determined. In the present work we used polarized macrophages derived from mice with macrophage-specific deficiency of TRPC3 to examine the expression level of ER stress markers and the activation status of some typical mediators of macrophage apoptosis. We found that the reduced susceptibility of TRPC3-deficient M1 macrophages to ER stress-induced apoptosis correlates with an impaired unfolded protein response (UPR), reduced mitochondrion-dependent apoptosis, and reduced activation of the proapoptotic molecules calmodulin-dependent protein kinase II and signal transducer and activator of transcription 1. Notably, none of these pathways was altered in TRPC3-deficient M2 macrophages. These findings show for the first time an obligatory requirement for a member of the TRPC family of cation channels in ER stress-induced apoptosis in macrophages, underscoring a rather selective role of the TRPC3 channel on mechanisms related to the UPR signaling in M1 macrophages.