Novel Role of the Transient Receptor Potential Canonical 3 (TRPC3) Channel in Macrophage Apoptosis: Implications in Atherosclerosi

Atherosclerosis is the main cause of death due to cardiovascular disease in western societies with macrophages playing important roles in all stages of lesion development. Macrophages exhibit heterogeneity in plaques with M1 (inflammatory/classically activated) and M2 (anti-inflammatory/alternatively activated) phenotypes being found in both human and murine atherosclerotic lesions. Macrophage apoptosis determines the lesion progression, fate and endoplasmic reticulum (ER) stress is one of its major triggers. In the advanced lesions, macrophage apoptosis coupled with defective phagocytic clearance (a process called efferocytosis) leads to secondary necrosis, culminating in formation of areas of necrosis, key determinant of plaques to rupture. Therefore, identifying mechanisms regulating macrophage apoptosis represents an attractive area for novel therapeutic opportunities. The Transient Receptor Potential Canonical 3 (TRPC3) channel is a non-voltage gated; non-selective cation channel endowed with significant constitutive activity and is prominently expressed in central nervous and cardiovascular systems. Studies from our laboratory showed that macrophage deficiency of Trpc3 resulted in impaired survival signaling and increased apoptosis of naive or 'M0' macrophages. As macrophage apoptosis influences lesion progression, we hypothesized that macrophage deficiency of Trpc3 would lead to increased apoptosis and necrosis in advanced atherosclerosis. Contrary to this, we found that advanced aortic root lesions of ApoE-/- mice (athero-prone background) transplanted with Trpc3 deficient bone marrow had reduced areas of necrosis and less apoptotic macrophages. These observations prompted us to speculate that TRPC3 might have a differential effect on apoptosis of naive (M0) vs. differentiated (M1 and M2) macrophages, latter phenotypes being predominant in atherosclerotic lesions. Also these studies made use of bone marrow donors which were global knockouts for Trpc3. Therefore the contributions from Trpc3 deficient non-myeloid cells to observed phenotype couldn't be ruled out and observations made were inconclusive. In this dissertation, we present a new mouse model generated in our laboratory, mice with macrophage specific loss of Trpc3 (MacTrpc3 -/- or MacTrpc3KO) to more specifically examine the role of macrophage Trpc3 in advanced atherosclerosis. We first investigated the impact of macrophage specific loss of Trpc3 on ER-stress induced apoptosis of M1 and M2 phenotypes. Remarkably, we found that macrophage specific loss of Trpc3 resulted in reduced ER--stress induced apoptosis of M1 macrophages with decreased activation of unfolded protein response signaling and other pro-apoptotic mediators downstream of ER stress. However none of these were altered in M2 cells. To further validate our in vitro findings in vivo, we next assessed the impact of macrophage deletion of Trpc3 on characteristics of advanced atherosclerotic lesions. Similar to our previous approach, low density lipoprotein receptor knockout mice (Ldlr-/-, another athero-prone background) were irradiated and reconstituted with Ldlr-/- or MacTrpc3 -/-/Ldlr-/- bone marrow and kept on high fat diet. Compared to controls, we found that advanced lesions of MacTrpc3-/- /Ldlr-/- mice had reduced macrophage apoptosis and plaque necrosis. We also found reduced number of apoptotic M1 but not M2 macrophages in the advanced lesions of MacTrpc3-/-/Ldlr-/- mice compared to controls. These findings confirmed our previous in vitro observations and provided evidence underscoring a pro-atherogenic role of macrophage Trpc3 in atherosclerosis.