Gsα Regulates Macrophage Foam Cell Formation During Atherosclerosis.

BACKGROUND: Many cardiovascular pathologies are induced by signaling through G-protein-coupled receptors via Gsα (G protein stimulatory α subunit) proteins. However, the specific cellular mechanisms that are driven by Gsα and contribute to the development of atherosclerosis remain unclear. METHODS: High-throughput screening involving data from single-cell and bulk sequencing were used to explore the expression of Gsα in atherosclerosis. The differentially expression and activity of Gsα were analyzed by immunofluorescence and cAMP measurements. Macrophage-specific Gsα knockout (Mac-GsαKO) mice were generated to study the effect on atherosclerosis. The role of Gsα was determined by transplanting bone marrow and performing assays for foam cell formation, Dil-ox-LDL (oxidized low-density lipoprotein) uptake, chromatin immunoprecipitation, and luciferase reporter assays. RESULTS: ScRNA-seq showed elevated Gnas in atherosclerotic mouse aorta's cholesterol metabolism macrophage cluster, while bulk sequencing confirmed increased GNAS expression in human plaque macrophage content. A significant upregulation of Gsα and active Gsα occurred in macrophages from human and mouse plaques. Ox-LDL could translocate Gsα from macrophage lipid rafts in short-term and promote Gnas transcription through ERK1/2 activation and C/EBPß phosphorylation via oxidative stress in long-term. Atherosclerotic lesions from Mac-GsαKO mice displayed decreased lipid deposition compared with those from control mice. Additionally, Gsα deficiency alleviated lipid uptake and foam cell formation. Mechanistically, Gsα increased the levels of cAMP and transcriptional activity of the cAMP response element binding protein, which resulted in increased expression of CD36 and SR-A1. In the translational experiments, inhibiting Gsα activation with suramin or cpGN13 reduced lipid uptake, foam cell formation, and the progression of atherosclerotic plaques in mice in vivo. CONCLUSIONS: Gsα activation is enhanced during atherosclerotic progression and increases lipid uptake and foam cell formation. The genetic or chemical inactivation of Gsα inhibit the development of atherosclerosis in mice, suggesting that drugs targeting Gsα may be useful in the treatment of atherosclerosis.

Endothelial G protein stimulatory α-subunit is a critical regulator of post-ischemic angiogenesis.

Post-ischemic angiogenesis is a vital pathophysiological process in diseases such as peripheral arterial disease (PAD), heart ischemia, and diabetic retinopathy. The molecular mechanisms of post-ischemic angiogenesis are complicated and not fully elucidated. The G protein stimulatory alpha subunit (Gsα) is essential for hormone-stimulated cyclic adenosine monophosphate (cAMP) production and is an important regulator for many physiological processes. In the present study, we investigated the role of endothelial Gsα in post-ischemic angiogenesis by generating adult mice with endothelial-specific Gsα deficiency (GsαECKO). GsαECKO mice had impaired blood flow recovery after hind limb ischemic injury, and reduced neovascularization in allograft transplanted tumors. Mechanically, Gsα could regulate the expression of angiogenic factor with G patch and FHA domains 1 (AGGF1) through cAMP/CREB pathway. AGGF1 plays a key role in angiogenesis and regulates endothelial cell proliferation as well as migration. Knockdown of CREB or mutation of the CRE site on the AGGF1 promoter led to reduced AGGF1 promoter activity. In addition, knockdown of AGGF1 reduced the proangiogenic effect of Gsα in endothelial cells, and overexpression of AGGF1 reversed the impaired angiogenesis in GsαECKO mice in vivo. The finding may prove useful in designing new therapeutic targets for treatments of post-ischemic angiogenesis-related diseases.

Stimulatory G-Protein α Subunit Modulates Endothelial Cell Permeability Through Regulation of Plasmalemma Vesicle-Associated Protein.

Endothelial cell leakage occurs in several diseases. Intracellular junctions and transcellular fashion are involved. The definite regulatory mechanism is complicated and not fully elucidated. The alpha subunit of the heterotrimeric G-stimulatory protein (Gsα) mediates receptor-stimulated production of cyclic adenosine monophosphate (cAMP). However, the role of Gsα in the endothelial barrier remains unclear. In this study, mice with knockout of endothelial-specific Gsα (GsαECKO) were generated by crossbreeding Gsαflox/flox mice with Cdh5-CreERT2 transgenic mice, induced in adult mice by tamoxifen treatment. GsαECKO mice displayed phenotypes of edema, anemia, hypoproteinemia and hyperlipoproteinemia, which indicates impaired microvascular permeability. Mechanistically, Gsα deficiency reduces the level of endothelial plasmalemma vesicle-associated protein (PLVAP). In addition, overexpression of Gsα increased phosphorylation of cAMP response element-binding protein (CREB) as well as the mRNA and protein levels of PLVAP. CREB could bind to the CRE site of PLVAP promoter and regulate its expression. Thus, Gsα might regulate endothelial permeability via cAMP/CREB-mediated PLVAP expression.

Smooth muscle-specific Gsα deletion exaggerates angiotensin II-induced abdominal aortic aneurysm formation in mice in vivo.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease without an effective pharmaceutical treatment. Genetic studies have proved the involvement of smooth muscle phenotype switch in the development of AAA. The alpha subunit of the heterotrimeric G stimulatory protein (Gsα) mediates receptor-stimulated production of cyclic adenosine monophosphate (cAMP). However, the role of smooth muscle Gsα in AAA formation remains unknown. APPROACH AND RESULTS: In this study, mice with knockout of smooth muscle-specific Gsα (GsαSMKO) were generated by cross-breeding Gsαflox/flox mice with SM22-CreERT2 transgenic mice, induced in adult mice by tamoxifen treatment. Gsα deficiency induced a smooth muscle phenotype switch from a contractile to a synthetic state. Mechanically, Gsα deletion reduced cAMP level and increased the level of human antigen R (HuR), which binds with the adenylate uridylate-rich elements of the 3' untranslated region of Krüppel-like factor 4 (KLF4) mRNA, thereby increasing the stability of KLF4. Moreover, genetic knockdown of HuR or KLF4 rescued the phenotype switch in Gsα-deficient smooth muscle cells. Furthermore, with acute infusion of angiotensin II, the incidence of AAA was markedly higher in ApoE-/-/GsαSMKO than ApoE-/-/Gsαflox/flox mice and induced increased elastic lamina degradation and aortic expansion. Finally, the levels of Gsα and SM α-actin were significantly lower while those of HuR and KLF4 were higher in human AAA samples than adjacent nonaneurysmal aortic sections. CONCLUSIONS: Gsα may play a protective role in AAA formation by regulating the smooth muscle phenotype switch and could be a potential therapeutic target for AAA disease.

Smooth muscle-specific LKB1 deletion exaggerates angiotensin II-induced abdominal aortic aneurysm in mice.

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease without an effective pharmaceutical treatment. Liver kinase B1 (LKB1), a tumor suppressor, is a central regulator of cell polarity and energy homeostasis. However, the role of LKB1 in the development of AAA has not been explored. In this study, mice with knockout of smooth muscle-specific LKB1 (LKB1SMKO) were generated by cross-breeding LKB1flox/flox mice with SM22-CreERT2 transgenic mice and induced in adult mice by tamoxifen treatment. LKB1 deficiency increased the expression of matrix metalloproteinase 2 (MMP-2), which was inhibited by LKB1 overexpression. Mechanistically, LKB1 could bind to the MMP-2 transcription factor, specificity protein 1 (Sp1), thereby reducing the binding of Sp1 to the MMP-2 promoter to inhibit MMP-2 expression. LKB1 expression was significantly reduced in abdominal aortas of the mouse AAA model. Moreover, smooth muscle-specific LKB1 deletion exaggerated angiotensin II-induced AAA formation accompanied by increased AAA incidence and aortic expansion. Finally, LKB1 level was significantly lower and MMP-2 level higher in human AAA samples than adjacent nonaneurysmal aortic sections. Thus, these results suggest that LKB1 may play a protective role in AAA formation by inhibiting MMP-2 expression and could be a potential therapeutic target for AAA disease.

Sp1 Plays an Important Role in Vascular Calcification Both In Vivo and In Vitro.

BACKGROUND: Vascular calcification and increased cardiovascular morbidity and mortality are closely related in patients with end-stage renal disease and diabetes mellitus. Specific protein 1 (Sp1) is a transactivation molecule that plays a crucial role in the regulation of apoptosis, fibrosis, angiogenesis, and other pathological disorders. There is evidence that specific protein 1 (Sp1) directly stimulates the transcription of bone morphogenetic protein 2 (BMP2) and that BMP2 plays a key role in the calcification process in the BMP2-expressing F9 cell model system. Here, we investigated whether Sp1 plays an important role in vascular calcification and its potential regulatory mechanism in vascular calcification. METHODS AND RESULTS: In this study, vascular calcification was induced in male Wistar rats by administration of nicotine (25 mg/kg) and vitamin D3 (300 000 IU/kg). These rats were randomly selected for treatment with adenovirus harboring Sp1 knockdown gene or empty virus. The mechanism of Sp1 in vascular smooth muscle cells cultured in high phosphate medium was studied. Based on our findings, the Sp1 gene silencing or inhibition improved calcium deposition, which was partly achieved by inhibiting phenotype switch, apoptosis, and matrix vesicle release of vascular smooth muscle cells. Moreover, Sp1 can activate BMP2 transcription by binding to the Sp1-binding element of the BMP2 promoter. CONCLUSIONS: Overall, elevated Sp1 exerts a pro-apoptotic effect, promoting BMP2 transcription and further accumulating vascular calcification. Proper and timely regulation of Sp1 expression may be a potential strategy for treatment of aging, end-stage renal disease, and diabetic-related macrovascular disease treatment.

Heterotrimeric G Stimulatory Protein α Subunit Is Required for Intestinal Smooth Muscle Contraction in Mice.

BACKGROUND & AIMS: The α subunit of the heterotrimeric G stimulatory protein (Gsa), encoded by the guanine nucleotide binding protein, α-stimulating gene (Gnas, in mice), is expressed ubiquitously and mediates receptor-stimulated production of cyclic adenosine monophosphate and activation of the protein kinase A signaling pathway. We investigated the roles of Gsa in vivo in smooth muscle cells of mice. METHODS: We performed studies of mice with Cre recombinase-mediated disruption of Gnas in smooth muscle cells (GsaSMKO and SM22-CreERT2, induced in adult mice by tamoxifen). Intestinal tissues were collected for histologic, biochemical, molecular, cell biology, and physiology analyses. Intestinal function was assessed in mice using the whole-gut transit time test. We compared gene expression patterns of intestinal smooth muscle from mice with vs without disruption of Gnas. Biopsy specimens from ileum of patients with chronic intestinal pseudo-obstruction and age-matched control biopsies were analyzed by immunohistochemistry. RESULTS: Disruption of Gnas in smooth muscle of mice reduced intestinal motility and led to death within 4 weeks. Tamoxifen-induced disruption of Gnas in adult mice impaired contraction of intestinal smooth muscle and peristalsis. More than 80% of these died within 3 months of tamoxifen exposure, with features of intestinal pseudo-obstruction characterized by chronic intestinal dilation and dysmotility. Gsa deficiency reduced intestinal levels of cyclic adenosine monophosphate and transcriptional activity of the cyclic adenosine monophosphate response element binding protein 1 (CREB1); this resulted in decreased expression of the forkhead box F1 gene (Foxf1) and protein, and contractile proteins, such as myosin heavy chain 11; actin, α2, smooth muscle, aorta; calponin 1; and myosin light chain kinase. We found decreased levels of Gsa, FOXF1, CREB1, and phosphorylated CREB1 proteins in intestinal muscle layers of patients with chronic intestinal pseudo-obstruction, compared with tissues from controls. CONCLUSIONS: Gsa is required for intestinal smooth muscle contraction in mice, and its levels are reduced in ileum biopsies of patients with chronic intestinal pseudo-obstruction. Mice with disruption of Gnas might be used to study human chronic intestinal pseudo-obstruction.