Peptide Vaccine Against ADAMTS-7 Ameliorates Atherosclerosis and Postinjury Neointima Hyperplasia.

BACKGROUND: The metalloprotease ADAMTS-7 (a disintegrin and metalloproteinase with thrombospondin type 1 motif 7) is a novel locus associated with human coronary atherosclerosis. ADAMTS-7 deletion protects against atherosclerosis and vascular restenosis in rodents. METHODS: We designed 3 potential vaccines consisting of distinct B cell epitopic peptides derived from ADAMTS-7 and conjugated with the carrier protein KLH (keyhole limpet hemocyanin) as well as aluminum hydroxide as an adjuvant. Arterial ligation or wire injury was used to induce neointima in mice, whereas ApoE-/- and LDLR-/- (LDLR [low-density lipoprotein receptor]) mice fed a high-fat diet were applied to assess atherosclerosis. In addition, coronary stent implantation was performed on vaccine-immunized Bama miniature pigs, followed by optical coherence tomography to evaluate coronary intimal hyperplasia. RESULTS: A vaccine, ATS7vac, was screened out from 3 candidates to effectively inhibit intimal thickening in murine carotid artery ligation models after vaccination. As well, immunization with ATS7vac alleviated neointima formation in murine wire injury models and mitigated atherosclerotic lesions in both hyperlipidemic ApoE-/- and LDLR-/- mice without lowering lipid levels. Preclinically, ATS7vac markedly impeded intimal hyperplasia in swine stented coronary arteries, but without significant immune-related organ injuries. Mechanistically, ATS7vac vaccination produced specific antibodies against ADAMTS-7, which markedly repressed ADAMTS-7-mediated COMP (cartilage oligomeric matrix protein) and TSP-1 (thrombospondin-1) degradation and subsequently inhibited vascular smooth muscle cell migration but promoted re-endothelialization. CONCLUSIONS: ATS7vac is a novel atherosclerosis vaccine that also alleviates in-stent restenosis. The application of ATS7vac would be a complementary therapeutic avenue to the current lipid-lowering strategy for atherosclerotic disease.

Nidogen-2 is a Novel Endogenous Ligand of LGR4 to Inhibit Vascular Calcification.

BACKGROUND: Vascular calcification is closely related to the all-cause mortality of cardiovascular events. Basement membrane protein nidogen-2 is a key component of the vascular extracellular matrix microenvironment and we recently found it is pivotal for the maintenance of contractile phenotype in vascular smooth muscle cells (VSMCs). However, whether nidogen-2 is involved in VSMCs osteochondrogenic transition and vascular calcification remains unclear. METHODS: VSMCs was treated with high-phosphate to study VSMC calcification in vitro. Three different mice models (5/6 nephrectomy-induced chronic renal failure, cholecalciferol-overload, and periadventitially administered with CaCl2) were used to study vascular calcification in vivo. Membrane protein interactome, coimmunoprecipitation, flow cytometric binding assay, surface plasmon resonance, G protein signaling, VSMCs calcium assays were performed to clarify the phenotype and elucidate the molecular mechanisms. RESULTS: Nidogen-2 protein levels were significantly reduced in calcified VSMCs and aortas from mice in different vascular calcification model. Nidogen-2 deficiency exacerbated high-phosphate-induced VSMC calcification, whereas the addition of purified nidogen-2 protein markedly alleviated VSMC calcification in vitro. Nidogen-2-/- mice exhibited aggravated aorta calcification compared to wild-type (WT) mice in response to 5/6 nephrectomy, cholecalciferol-overload, and CaCl2 administration. Further unbiased coimmunoprecipitation and interactome analysis of purified nidogen-2 and membrane protein in VSMCs revealed that nidogen-2 directly binds to LGR4 (leucine-rich repeat G-protein-coupled receptor 4) with KD value 26.77 nM. LGR4 deficiency in VSMCs in vitro or in vivo abolished the protective effect of nidogen-2 on vascular calcification. Of interest, nidogen-2 biased activated LGR4-Gαq-PKCα (protein kinase Cα)-AMPKα1 (AMP-activated protein kinase α1) signaling to counteract VSMCs osteogenic transition and mineralization. CONCLUSIONS: Nidogen-2 is a novel endogenous ligand of LGR4 that biased activated Gαq- PKCα-AMPKα1 signaling and inhibited vascular calcification.

PHB2 Maintains the Contractile Phenotype of VSMCs by Counteracting PKM2 Splicing.

BACKGROUND: Phenotypic transition of vascular smooth muscle cells (VSMCs) accounts for the pathogenesis of a variety of vascular diseases during the early stage. Recent studies indicate the metabolic reprogramming may be involved in VSMC phenotypic transition. However, the definite molecules that link energy metabolism to distinct VSMC phenotype remain elusive. METHODS: A carotid artery injury model was used to study postinjury neointima formation as well as VSMC phenotypic transition in vivo. RNA-seq analysis, cell migration assay, collagen gel contraction assay, wire myography assay, immunoblotting, protein interactome analysis, co-immunoprecipitation, and mammalian 2-hybrid assay were performed to clarify the phenotype and elucidate the molecular mechanisms. RESULTS: We collected cell energy-regulating genes by using Gene Ontology annotation and applied RNA-Seq analysis of transforming growth factor-ß or platelet-derived growth factor BB stimulated VSMCs. Six candidate genes were overlapped from energy metabolism-related genes and genes reciprocally upregulated by transforming growth factor-ß and downregulated by platelet-derived growth factor BB. Among them, prohibitin 2 has been reported to regulate mitochondrial oxidative phosphorylation. Indeed, prohibitin 2-deficient VSMCs lost the contractile phenotype as evidenced by reduced contractile proteins. Consistently, Phb2SMCKO mice were more susceptible to postinjury VSMC proliferation and neointima formation compared with Phb2flox/flox mice. Further protein interactome analysis, co-immunoprecipitation, and mammalian 2-hybrid assay revealed that prohibitin 2, through its C-terminus, directly interacts with hnRNPA1, a key modulator of pyruvate kinase M1/2 (PKM) mRNA splicing that promotes PKM2 expression and glycolysis. Prohibitin 2 deficiency facilitated PKM1/2 mRNA splicing and reversion from PKM1 to PKM2, and enhanced glycolysis in VSMCs. Blocking prohibitin 2-hnRNPA1 interaction resulted in increased PKM2 expression, enhanced glycolysis, repressed contractile marker genes expression in VSMCs, as well as aggravated postinjury neointima formation in vivo. CONCLUSIONS: Prohibitin 2 maintains VSMC contractile phenotype by interacting with hnRNPA1 to counteract hnRNPA1-mediated PKM alternative splicing and glucose metabolic reprogramming.

ADAMTS7-Mediated Complement Factor H Degradation Potentiates Complement Activation to Contributing to Renal Injuries.

BACKGROUND: The dysfunction of complement factor H (CFH), the main soluble complement negative regulator, potentiates various complement-induced renal injuries. However, insights into the underlying mechanism of CFH dysfunction remain limited. In this study, we investigated whether extracellular protease-mediated degradation accounts for CFH dysfunction in complement-mediated renal injuries. METHODS: An unbiased interactome of lupus mice kidneys identified CFH-binding protease. In vitro cleavage assay clarified CFH degradation. Pristane-induced SLE or renal ischemia-reperfusion (I/R) injury models were used in wild-type and ADAMTS7-/- mice. RESULTS: We identified the metalloprotease ADAMTS7 as a CFH-binding protein in lupus kidneys. Moreover, the upregulation of ADAMTS7 correlated with CFH reduction in both lupus mice and patients. Mechanistically, ADAMTS7 is directly bound to CFH complement control protein (CCP) 1-4 domain and degraded CCP 1-7 domain through multiple cleavages. In mice with lupus nephritis or renal I/R injury, ADAMTS7 deficiency alleviated complement activation and related renal pathologies, but without affecting complement-mediated bactericidal activity. Adeno-associated virus-mediated CFH silencing compromised these protective effects of ADAMTS7 knockout against complement-mediated renal injuries in vivo. CONCLUSION: ADAMTS7-mediated CFH degradation potentiates complement activation and related renal injuries. ADAMTS7 would be a promising anticomplement therapeutic target that does not increase bacterial infection risk.

X-box binding protein 1-mediated COL4A1s secretion regulates communication between vascular smooth muscle and stem/progenitor cells.

Vascular smooth muscle cells (VSMCs) contribute to the deposition of extracellular matrix proteins (ECMs), including Type IV collagen, in the vessel wall. ECMs coordinate communication among different cell types, but mechanisms underlying this communication remain unclear. Our previous studies have demonstrated that X-box binding protein 1 (XBP1) is activated and contributes to VSMC phenotypic transition in response to vascular injury. In this study, we investigated the participation of XBP1 in the communication between VSMCs and vascular progenitor cells (VPCs). Immunofluorescence and immunohistology staining revealed that Xbp1 gene was essential for type IV collagen alpha 1 (COL4A1) expression during mouse embryonic development and vessel wall ECM deposition and stem cell antigen 1-positive (Sca1+)-VPC recruitment in response to vascular injury. The Western blot analysis elucidated an Xbp1 gene dose-dependent effect on COL4A1 expression and that the spliced XBP1 protein (XBP1s) increased protease-mediated COL4A1 degradation as revealed by Zymography. RT-PCR analysis revealed that XBP1s in VSMCs not only upregulated COL4A1/2 transcription but also induced the occurrence of a novel transcript variant, soluble type IV collagen alpha 1 (COL4A1s), in which the front part of exon 4 is joined with the rear part of exon 42. Chromatin-immunoprecipitation, DNA/protein pulldown and in vitro transcription demonstrated that XBP1s binds to exon 4 and exon 42, directing the transcription from exon 4 to exon 42. This leads to transcription complex bypassing the internal sequences, producing a shortened COL4A1s protein that increased Sca1+-VPC migration. Taken together, these results suggest that activated VSMCs may recruit Sca1+-VPCs via XBP1s-mediated COL4A1s secretion, leading to vascular injury repair or neointima formation.

Nidogen-2 Maintains the Contractile Phenotype of Vascular Smooth Muscle Cells and Prevents Neointima Formation via Bridging Jagged1-Notch3 Signaling.

BACKGROUND: How the extracellular matrix (ECM) microenvironment modulates the contractile phenotype of vascular smooth muscle cells (VSMCs) and confers vascular homeostasis remains elusive. METHODS: To explore the key ECM proteins in the maintenance of the contractile phenotype of VSMCs, we applied protein-protein interaction network analysis to explore novel ECM proteins associated with the VSMC phenotype. By combining in vitro and in vivo genetic mice vascular injury models, we identified nidogen-2, a basement membrane glycoprotein, as a key ECM protein for maintenance of vascular smooth muscle cell identity. RESULTS: We collected a VSMC phenotype-related gene dataset by using Gene Ontology annotation combined with a literature search. A computational analysis of protein-protein interactions between ECM protein genes and the genes from the VSMC phenotype-related gene dataset revealed the candidate gene nidogen-2, a basement membrane glycoprotein involved in regulation of the VSMC phenotype. Indeed, nidogen-2-deficient VSMCs exhibited loss of contractile phenotype in vitro, and compared with wild-type mice, nidogen-2-/- mice showed aggravated post-wire injury neointima formation of carotid arteries. Further bioinformatics analysis, coimmunoprecipitation assays, and luciferase assays revealed that nidogen-2 specifically interacted with Jagged1, a conventional Notch ligand. Nidogen-2 maintained the VSMC contractile phenotype via Jagged1-Notch3 signaling but not Notch1 or Notch2 signaling. Nidogen-2 enhanced Jagged1 and Notch3 interaction and subsequent Notch3 activation. Reciprocally, Jagged1 and Notch3 interaction, signaling activation, and Jagged1-triggered VSMC differentiation were significantly repressed in nidogen-2-deficient VSMCs. In accordance, the suppressive effect of Jagged1 overexpression on neointima formation was attenuated in nidogen-2-/- mice compared with wild-type mice. CONCLUSIONS: Nidogen-2 maintains the contractile phenotype of VSMCs through Jagged1-Notch3 signaling in vitro and in vivo. Nidogen-2 is required for Jagged1-Notch3 signaling.

Extracellular matrix dynamics in vascular remodeling.

Vascular remodeling is the adaptive response to various physiological and pathophysiological alterations that are closely related to aging and vascular diseases. Understanding the mechanistic regulation of vascular remodeling may be favorable for discovering potential therapeutic targets and strategies. The extracellular matrix (ECM), including matrix proteins and their degradative metalloproteases, serves as the main component of the microenvironment and exhibits dynamic changes during vascular remodeling. This process involves mainly the altered composition of matrix proteins, metalloprotease-mediated degradation, posttranslational modification of ECM proteins, and altered topographical features of the ECM. To date, adequate studies have demonstrated that ECM dynamics also play a critical role in vascular remodeling in various diseases. Here, we review these related studies, summarize how ECM dynamics control vascular remodeling, and further indicate potential diagnostic biomarkers and therapeutic targets in the ECM for corresponding vascular diseases.

Cartilage oligomeric matrix protein is a novel notch ligand driving embryonic stem cell differentiation towards the smooth muscle lineage.

Cartilage oligomeric matrix protein (COMP), a protective component of vascular extracellular matrix (ECM), maintains the homeostasis of mature vascular smooth muscle cells (VSMCs). However, whether COMP modulates the differentiation of stem cells towards the smooth muscle lineage is still elusive. Firstly, purified mouse COMP directly induced mouse embryonic stem cell (ESC) differentiation into VSMCs both in vitro and in vivo, while the silencing of endogenous COMP markedly inhibited ESC-VSMC differentiation. RNA-Sequencing revealed that Notch signaling was significantly activated by COMP during ESC-VSMC differentiation, whereas the inhibition of Notch signaling attenuated COMP-directed ESC-VSMC differentiation. Furthermore, COMP deficiency inhibited Notch activation and VSMC differentiation in mice. Through silencing distinct Notch receptors, we identified that Notch1 mainly mediated COMP-initiated ESC-VSMC differentiation. Mechanistically, COMP N-terminus directly interacted with the EGF11-12 domain of Notch1 and activated Notch1 signaling, as evidenced by co-immunoprecipitation and mammalian two-hybrid assay. In conclusion, COMP served as a potential ligand of Notch1, thereby driving ESC-VSMC differentiation.

Targeting macrophage TFEB-14-3-3 epsilon Interface by naringenin inhibits abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a lethal cardiovascular disease, and there is no proven drug treatment for this condition. In this study, by using the Connectivity Map (CMap) approach, we explored naringenin, a naturally occurring citrus flavonoid, as a putative agent for inhibiting AAA. We then validated the prediction with two independent mouse models of AAA, calcium phosphate (CaPO4)-induced C57BL/6J mice and angiotensin II-infused ApoE-/- mice. Naringenin effectively blocked the formation of AAAs and the progression of established AAAs. Transcription factor EB (TFEB) is the master regulator of lysosome biogenesis. Intriguingly, the protective role of naringenin on AAA was abolished by macrophage-specific TFEB depletion in mice. Unbiased interactomics, combined with isothermal titration calorimetry (ITC) and cellular thermal shift assays (CETSAs), further revealed that naringenin is directly bound to 14-3-3 epsilon blocked the TFEB-14-3-3 epsilon interaction, and therefore promoted TFEB nuclear translocation and activation. On one hand, naringenin activated lysosome-dependent inhibition of the NLRP3 inflammasome and repressed aneurysmal inflammation. On the other hand, naringenin induced TFEB-dependent transcriptional activation of GATA3, IRF4, and STAT6 and therefore promoted reparative M2 macrophage polarization. In summary, naturally derived naringenin or macrophage TFEB activation shows promising efficacy for the treatment of AAA.

Cartilage oligomeric matrix protein is an endogenous ß-arrestin-2-selective allosteric modulator of AT1 receptor counteracting vascular injury.

Compelling evidence has revealed that biased activation of G protein-coupled receptor (GPCR) signaling, including angiotensin II (AngII) receptor type 1 (AT1) signaling, plays pivotal roles in vascular homeostasis and injury, but whether a clinically relevant endogenous biased antagonism of AT1 signaling exists under physiological and pathophysiological conditions has not been clearly elucidated. Here, we show that an extracellular matrix protein, cartilage oligomeric matrix protein (COMP), acts as an endogenous allosteric biased modulator of the AT1 receptor and its deficiency is clinically associated with abdominal aortic aneurysm (AAA) development. COMP directly interacts with the extracellular N-terminus of the AT1 via its EGF domain and inhibits AT1-ß-arrestin-2 signaling, but not Gq or Gi signaling, in a selective manner through allosteric regulation of AT1 intracellular conformational states. COMP deficiency results in activation of AT1a-ß-arrestin-2 signaling and subsequent exclusive AAA formation in response to AngII infusion. AAAs in COMP-/- or ApoE-/- mice are rescued by AT1a or ß-arrestin-2 deficiency, or the application of a peptidomimetic mimicking the AT1-binding motif of COMP. Explorations of the endogenous biased antagonism of AT1 receptor or other GPCRs may reveal novel therapeutic strategies for cardiovascular diseases.

Autoantibodies against AT1 Receptor Contribute to Vascular Aging and Endothelial Cell Senescence.

Vascular aging predisposes the elderly to the progression of many aging-related vascular disorders and leads to deterioration of cardiovascular diseases (CVD). However, the underlying mechanisms have not been clearly elucidated. Agonistic autoantibodies against angiotensin II type 1 (AT1) receptor (AT1-AAs) have been demonstrated to be pro-inflammatory and contribute to the progression of atherosclerosis. However, the association between AT1-AAs and vascular aging has not been defined. Peripheral arterial disease (PAD) is an acknowledged vascular aging-related disease. In this study, AT1-AAs were detected in the sera of patients with PAD and the positive rate was 44.44% (n=63) vs. 17.46% in non-PAD volunteers (n=63). In addition, case-control analysis showed that AT1-AAs level was positively correlated with PAD. To reveal the causal relationship between AT1-AAs and vascular aging, an AT1-AAs-positive rat model was established by active immunization. The carotid pulse wave velocity was higher, and the aortic endothelium-dependent vasodilatation was attenuated significantly in the immunized rats. Morphological staining showed thickening of the aortic wall. Histological examination showed that levels of the senescent markers were increased in the aortic tissue, mostly located at the endothelium. In addition, purified AT1-AAs-IgGs from both the immunized rats and PAD patients induced premature senescence in cultured human umbilical vein endothelial cells. These effects were significantly blocked by the AT1 receptor blocker. Taken together, our study demonstrates that AT1-AAs contribute to the progression of vascular aging and induce EC senescence through AT1 receptor. AT1-AA is a novel biomarker of vascular aging and aging-related CVD that acts to accelerate EC senescence.

COMP-prohibitin 2 interaction maintains mitochondrial homeostasis and controls smooth muscle cell identity.

Vascular smooth muscle cells (VSMCs) are highly phenotypically plastic, and loss of the contractile phenotype in VSMCs has been recognized at the early onset of the pathology of a variety of vascular diseases. However, the endogenous regulatory mechanism to maintain contractile phenotype in VSMCs remains elusive. Moreover, little has been known about the role of the mitochondrial bioenergetics in terms of VSMC homeostasis. Herein, we asked if glycoprotein COMP (Cartilage oligomeric matrix protein) is involved in mitochondrial bioenergetics and therefore regulates VSMCs homeostasis. By using fluorescence assay, subcellular western blot and liquid chromatography tandem mass spectrometry analysis, we found that extracellular matrix protein COMP unexpectedly localized within mitochondria. Further mitochondrial transplantation revealed that both mitochondrial and non-mitochondrial COMP maintained VSMC identity. Moreover, microarray analysis revealed that COMP deficiency impaired mitochondrial oxidative phosphorylation in VSMCs. Further study confirmed that COMP deficiency caused mitochondrial oxidative phosphorylation dysfunction accompanied by morphological abnormality. Moreover, the interactome of mitochondrial COMP revealed that COMP interacted with prohibitin 2, and COMP-prohibitin 2 interaction maintained mitochondrial homeostasis. Additionally, disruption of COMP-prohibitin 2 interaction caused VSMC dedifferentiation in vitro and enhanced the neointima formation post rat carotid artery injury in vivo. In conclusion, COMP-prohibitin 2 interaction in mitochondria plays an important role in maintaining the contractile phenotype of VSMCs by regulating mitochondrial oxidative phosphorylation. Maintaining the homeostasis of mitochondrial respiration through COMP-prohibitin 2 interaction may shed light on prevention of vascular disease.