Preventing Cholesterol-Induced Perk (Protein Kinase RNA-Like Endoplasmic Reticulum Kinase) Signaling in Smooth Muscle Cells Blocks Atherosclerotic Plaque Formation

BACKGROUND: Vascular smooth muscle cells (SMCs) undergo complex phenotypic modulation with atherosclerotic plaque formation in hyperlipidemic mice, which is characterized by de-differentiation and heterogeneous increases in the expression of macrophage, fibroblast, osteogenic, and stem cell markers. An increase of cellular cholesterol in SMCs triggers similar phenotypic changes in vitro with exposure to free cholesterol due to cholesterol entering the endoplasmic reticulum, triggering endoplasmic reticulum stress and activating Perk (protein kinase RNA-like endoplasmic reticulum kinase) signaling. METHODS: We generated an SMC-specific RESULTS: SMC-specific deletion of Perk reduces atherosclerotic plaque formation in male hyperlipidemic mice by 80%. Single-cell transcriptomic data identify 2 clusters of modulated SMCs in hyperlipidemic mice, one of which is absent when CONCLUSIONS: Our results indicate that hypercholesterolemia drives both Perk-dependent and Perk-independent SMC modulation and that deficiency of Perk significantly blocks atherosclerotic plaque formation

TARGETING METABOLIC ALTERATIONS ASSOCIATED WITH SMOOTH MUSCLE α-ACTIN PATHOGENIC VARIANT ATTENUATES MOYAMOYA-LIKE CEREBROVASCULAR DISEASE

Heterozygous pathogenic variants in ACTA2, encoding smooth muscle α-actin (α-SMA), predispose to thoracic aortic aneurysms and dissections. De novo missense variants disrupting ACTA2 arginine 179 (p.Arg179) cause a multisystemic disease termed smooth muscle dysfunction syndrome (SMDS), which is characterized by early onset thoracic aortic disease and moyamoya disease-like (MMD) cerebrovascular disease. The MMD-like cerebrovascular disease in SMDS patients is marked by bilateral steno-occlusive lesions in the distal internal carotid arteries (ICAs) and their branches. To study the molecular mechanisms that underlie the ACTA2 p.Arg179 variants, a smooth muscle-specific Cre-lox knock-in mouse model of the heterozygous Acta2 R179C variant, termed the Acta2SMC-R179C/+ mouse model, was generated. Acta2SMC-R179C/+ mice exhibit mild hypotension, but do not have spontaneous aortic disease, strokes, or death up to two years of age. In the Acta2SMC-R179C/+ mouse model, 67% of the smooth muscle cells (SMCs) in the vasculature express the heterozygous Acta2 R179C mutation. However, when SMCs are explanted from Acta2SMC-R179C/+ mouse aortas, the population is completely heterozygous for the mutation. Here, I show that Acta2SMC-R179C/+ SMCs are not fully differentiated and proliferate and migrate faster than wild-type (WT) SMCs. Metabolic profiling identified that the Acta2SMC-R179C/+ SMCs have increased glycolytic flux and decreased oxidative phosphorylation (OXPHOS), which is associated with reduced mitochondrial DNA and complex I activity, reflecting decreased electron transport chain activity. Nicotinamide riboside (NR), a NAD+ analogue, altered this metabolic profile. NR decreased glycolysis and increased OXPHOS by enhancing complex I activity without altering mitochondrial mass in Acta2SMC-R179C/+ SMCs. Furthermore, NR increased differentiation and decreased migration in Acta2SMC-R179C/+ SMCs. To determine how phenotypic changes in Acta2SMC-R179C/+ SMCs contribute to cerebrovascular disease, left carotid artery ligation (LCAL) was performed in these mice. One-fifth of Acta2SMC-R179C/+ mice die immediately post-ligation due to ischemic strokes, whereas no WT mice died. The surviving mutant mice have persistent intraluminal lesions containing SMCs that resemble MMD lesions, which is consistent with increased migration observed in mutant SMCs, along with medial thinning and an enlarged lumen area proximal to the ligation site. In contrast, the WT mice show nearly patent lumens with medial thickening. Additionally, Acta2SMC-R179C/+ mice display aberrant vascular remodeling consisting of increased neovascularization surrounding the left carotid artery, augmented leptomeningeal collateral remodeling, and greater post-occlusion stenosis in the large intracranial arteries when compared to the WT mice. To establish whether NR treatment alters outcomes in LCAL-injured Acta2SMC-R179C/+ mice, mice were administered NR every other day beginning five days prior to the ligation injury. NR treatment prevented deaths post-ligation and improved the vascular remodeling in the mutant mice. Specifically, NR partially resolved the intraluminal left carotid artery lesions, reduced neovascularization surrounding the left carotid artery, attenuated leptomeningeal collateral remodeling, and increased patency of the large intracranial arteries distal to the ligation. Together, these results establish a novel role for glycolytic metabolism in driving vascular occlusive disease. These results also highlight the potential of increasing mitochondrial metabolism in SMCs to restore a differentiated and quiescent phenotype and attenuate MMD-like cerebrovascular occlusive lesions to prevent ischemic strokes in patients with SMDS

Expanding ACTA2 genotypes with corresponding phenotypes overlapping with smooth muscle dysfunction syndrome

Pathogenic variants in ACTA2, encoding smooth muscle α-actin, predispose to thoracic aortic aneurysms and dissections. ACTA2 variants altering arginine 179 predispose to a more severe, multisystemic disease termed smooth muscle dysfunction syndrome (SMDS; OMIM 613834). Vascular complications of SMDS include patent ductus arteriosus (PDA) or aortopulmonary window, early-onset thoracic aortic disease (TAD), moyamoya-like cerebrovascular disease, and primary pulmonary hypertension. Patients also have dysfunction of other smooth muscle-dependent systems, including congenital mydriasis, hypotonic bladder, and gut hypoperistalsis. Here, we describe five patients with novel heterozygous ACTA2 missense variants, p.Arg179Gly, p.Met46Arg, p.Thr204Ile, p.Arg39Cys, and p.Ile66Asn, who have clinical complications that align or overlap with SMDS. Patients with the ACTA2 p.Arg179Gly and p.Thr204Ile variants display classic features of SMDS. The patient with the ACTA2 p.Met46Arg variant exhibits exclusively vascular complications of SMDS, including early-onset TAD, PDA, and moyamoya-like cerebrovascular disease. The patient with the ACTA2 p.Ile66Asn variant has an unusual vascular complication, a large fusiform internal carotid artery aneurysm. The patient with the ACTA2 p.Arg39Cys variant has pulmonary, gastrointestinal, and genitourinary complications of SMDS but no vascular manifestations. Identifying pathogenic ACTA2 variants associated with features of SMDS is critical for aggressive surveillance and management of vascular and nonvascular complications and delineating the molecular pathogenesis of SMDS.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/173096/1/ajmga62775.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/173096/2/ajmga62775_am.pd

The transcriptional regulation of the human angiotensinogen gene after high-fat diet is haplotype-dependent: Novel insights into the gene-regulatory networks and implications for human hypertension

<div><p>Single nucleotide polymorphisms (SNPs) in the human angiotensinogen (hAGT) gene may modulate its transcription and affect the regulation of blood pressure via activation of the renin-angiotensin aldosterone system (RAAS). In this regard, we have identified polymorphisms in the 2.5 Kb promoter of the hAGT gene that form two haplotype (Hap) blocks: -6A/G (-1670A/G, -1562C/T, -1561T/C) and -217A/G (-532T/C, -793A/G, -1074T/C & -1178G/A). hAGT gene with Hap -6A/-217A (Hap I) is associated with increased blood pressure whereas, Hap -6G/-217G (Hap II) is associated with normal blood pressure in human subjects. Since RAAS over activity contributes to hypertension in obesity, we have made transgenic mice (TG) containing either Hap I or Hap II of the hAGT gene to understand the role of obesity on its transcriptional regulation. Although, a high-fat diet (60% Kcal from fat, 12 weeks) elevates hAGT and mAGT regardless of haplotype, this effect is significantly (p<0.05) accentuated in Hap I mice, in both adipose and liver tissues. Chromatin Immuno- precipitation (ChIP) assay shows an increased binding of transcription factors including, GR, CEBPβ and STAT3 to the chromatin of the Hap I TG mice after high-fat diet as compared to Hap II TG mice (p<0.05). Differential plasma levels of hAGT in Hap II and I mice, after high-fat diet, further corroborate the variable transcriptional regulation of the hAGT, governed by gene-haplotypes. Taken together, our results show that SNPs in the Hap-I of the hAGT gene promote high-fat diet-induced binding of transcription factors GR, CEBP-β and STAT3, which lead to elevated expression of the hAGT gene in hepatic and adipose tissues.</p></div

ChIP assay on the -217 and -1329 regions of the hAGT gene from the chromatin obtained from adipose tissue of TG mice after HFD.

<p>ChIP assay was performed by PCR amplification of the immunoprecipitated DNA in the presence of antibodies against GR (a), CEBPβ (b) and STAT3 (c), input DNA (d), IgG (e), and nonspecific primers (NS) (f). Immunoprecipitated DNA was used to amplify nucleotide sequence encompassing either -217 region (Fig 4A) or -1329 region (Fig 4B) of the hAGT gene as described in “Materials and Methods.” Quantitation of GR, CEBPβ and STAT3 -enriched DNA, relative to input, at the -217 region or -1329 region of the hAGT gene was performed by Q-PCR. Result shows a significant increase in the HFD-induced GR, CEBPβ and STAT3 binding in TG mice with Hap I as compared to Hap II. *, p≤0.05 <i>versus</i> Hap II with HFD. Results are shown as mean ± S.E. (n = 4). A.U., arbitrary units.</p

TG-mice with Hap I have increased expression of the hAGT gene after HFD, as compared with Hap II.

<p>Human AGT expression is significantly elevated after HFD in TG mice with Hap I than Hap II, in adipose and liver tissues. Change in mRNA expression of the hAGT gene after 12 weeks of HFD, as compared to baseline CD, in adipose (A) and liver tissue (B). mAGT expression in adipose (C) and liver tissue (D) of TG mice fed with control diet (CD) or HFD in both haplotypes. mRNA was determined by quantitative RT-PCR analysis. Results are shown as mean ±SEM (error bars) from n = 4 per group. *p ≤ 0.05 <i>versus</i> Hap II with HFD; ^# p ≤0.05 <i>versus</i> CD in both Hap I & Hap II.</p

Expression of the transcription factors associated with the regulation of the hAGT, with or without HFD.

<p>Expression of transcription factors, GR, CEBPβ, and STAT3 with or without HFD in adipose (A) and liver (B) tissue. Expression of mRNA was calculated for the HFD and CD group and normalized by the respective GAPDH values. Results are shown as mean ± S.E. (error bars) (n = 4). *p ≤0.05 versus Hap I & Hap II with CD.</p

Effect of HFD on plasma level of hAGT in Hap I and Hap II TG mice.

<p>Plasma levels of hAGT in TG mice containing either Hap I or Hap II of the hAGT gene in CD or HFD fed TG mice. Results are shown as mean ± SEM from n = 4. *p≤0.05 versus Hap II with HFD; # p≤0.05 versus Hap II with CD.</p