Paricalcitol Has a Potent Anti-Inflammatory Effect in Rat Endothelial Denudation-Induced Intimal Hyperplasia.

Neointimal hyperplasia is the main cause of vascular graft failure in the medium term. Vitamin D receptor activation modulates the biology of vascular smooth muscle cells and has been reported to protect from neointimal hyperplasia following endothelial injury. However, the molecular mechanisms are poorly understood. We have now explored the impact of the selective vitamin D receptor activator, paricalcitol, on neointimal hyperplasia, following guidewire-induced endothelial cell injury in rats, and we have assessed the impact of paricalcitol or vehicle on the expression of key cell stress factors. Guidewire-induced endothelial cell injury caused neointimal hyperplasia and luminal stenosis and upregulated the expression of the growth factor growth/differentiation factor-15 (GDF-15), the cytokine receptor CD74, NFκB-inducing kinase (NIK, an upstream regulator of the proinflammatory transcription factor NFκB) and the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Immunohistochemistry confirmed the increased expression of the cellular proteins CD74 and NIK. Paricalcitol (administered in doses of 750 ng/kg of body weight, every other day) had a non-significant impact on neointimal hyperplasia and luminal stenosis. However, it significantly decreased GDF-15, CD74, NIK and MCP-1/CCL2 mRNA expression, which in paricalcitol-injured arteries remained within the levels found in control vehicle sham arteries. In conclusion, paricalcitol had a dramatic effect, suppressing the stress response to guidewire-induced endothelial cell injury, despite a limited impact on neointimal hyperplasia and luminal stenosis. This observation identifies novel molecular targets of paricalcitol in the vascular system, whose differential expression cannot be justified as a consequence of improved tissue injury.

LncRNA-LncDACH1 mediated phenotypic switching of smooth muscle cells during neointimal hyperplasia in male arteriovenous fistulas.

Arteriovenous fistulas (AVFs) are the most common vascular access points for hemodialysis (HD), but they have a high incidence of postoperative dysfunction, mainly due to excessive neointimal hyperplasia (NIH). Our previous studies have revealed a highly conserved LncRNA-LncDACH1 as an important regulator of cardiomyocyte and fibroblast proliferation. Herein, we find that LncDACH1 regulates NIH in AVF in male mice with conditional knockout of smooth muscle cell-specific LncDACH1 and in male mice model of AVF with LncDACH1 overexpression by adeno-associated virus. Mechanistically, silence of LncDACH1 activates p-AKT through promoting the expression of heat shock protein 90 (HSP90) and serine/arginine-rich splicing factor protein kinase 1 (SRPK1). Moreover, LncDACH1 is transcriptionally activated by transcription factor KLF9 that binds directly to the promoter region of the LncDACH1 gene. In this work, during AVF NIH, LncDACH1 is downregulated by KLF9 and promotes NIH through the HSP90/ SRPK1/ AKT signaling axis.

KLF13 promotes VSMCs phenotypic dedifferentiation by directly binding to the SM22α promoter.

Krüppel-like factor 13 (KLF13), a zinc finger transcription factor, is considered as a potential regulator of cardiomyocyte differentiation and proliferation during heart morphogenesis. However, its precise role in the dedifferentiation of vascular smooth muscle cells (VSMCs) during atherosclerosis and neointimal formation after injury remains poorly understood. In this study, we investigated the relationship between KLF13 and SM22α expression in normal and atherosclerotic plaques by bioanalysis, and observed a significant increase in KLF13 levels in the atherosclerotic plaques of both human patients and ApoE-/- mice. Knockdown of KLF13 was found to ameliorate intimal hyperplasia following carotid artery injury. Furthermore, we discovered that KLF13 directly binds to the SM22α promoter, leading to the phenotypic dedifferentiation of VSMCs. Remarkably, we observed a significant inhibition of platelet-derived growth factor BB-induced VSMCs dedifferentiation, proliferation, and migration when knocked down KLF13 in VSMCs. This inhibitory effect of KLF13 knockdown on VCMC function was, at least in part, mediated by the inactivation of p-AKT signaling in VSMCs. Overall, our findings shed light on a potential therapeutic target for treating atherosclerotic lesions and restenosis after vascular injury.

G6PD maintains the VSMC synthetic phenotype and accelerates vascular neointimal hyperplasia by inhibiting the VDAC1-Bax-mediated mitochondrial apoptosis pathway.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) plays an important role in vascular smooth muscle cell (VSMC) phenotypic switching, which is an early pathogenic event in various vascular remodeling diseases (VRDs). However, the underlying mechanism is not fully understood. METHODS: An IP‒LC‒MS/MS assay was conducted to identify new binding partners of G6PD involved in the regulation of VSMC phenotypic switching under platelet-derived growth factor-BB (PDGF-BB) stimulation. Co-IP, GST pull-down, and immunofluorescence colocalization were employed to clarify the interaction between G6PD and voltage-dependent anion-selective channel protein 1 (VDAC1). The molecular mechanisms involved were elucidated by examining the interaction between VDAC1 and apoptosis-related biomarkers, as well as the oligomerization state of VDAC1. RESULTS: The G6PD level was significantly elevated and positively correlated with the synthetic characteristics of VSMCs induced by PDGF-BB. We identified VDAC1 as a novel G6PD-interacting molecule essential for apoptosis. Specifically, the G6PD-NTD region was found to predominantly contribute to this interaction. G6PD promotes VSMC survival and accelerates vascular neointimal hyperplasia by inhibiting VSMC apoptosis. Mechanistically, G6PD interacts with VDAC1 upon stimulation with PDGF-BB. By competing with Bax for VDAC1 binding, G6PD reduces VDAC1 oligomerization and counteracts VDAC1-Bax-mediated apoptosis, thereby accelerating neointimal hyperplasia. CONCLUSION: Our study showed that the G6PD-VDAC1-Bax axis is a vital switch in VSMC apoptosis and is essential for VSMC phenotypic switching and neointimal hyperplasia, providing mechanistic insight into early VRDs.

Deficiency of smooth muscle cell ILF3 alleviates intimal hyperplasia via HMGB1 mRNA degradation-mediated regulation of the STAT3/DUSP16 axis.

Intimal hyperplasia is a complicated pathophysiological phenomenon attributable to in-stent restenosis, and the underlying mechanism remains unclear. Interleukin enhancer-binding factor 3 (ILF3), a double-stranded RNA-binding protein involved in regulating mRNA stability, has been recently demonstrated to assume a crucial role in cardiovascular disease; nevertheless, its impact on intimal hyperplasia remains unknown. In current study, we used samples of human restenotic arteries and rodent models of intimal hyperplasia, we found that vascular smooth muscle cell (VSMC) ILF3 expression was markedly elevated in human restenotic arteries and murine ligated carotid arteries. SMC-specific ILF3 knockout mice significantly suppressed injury induced neointimal formation. In vitro, platelet-derived growth factor type BB (PDGF-BB) treatment elevated the level of VSMC ILF3 in a dose- and time-dependent manner. ILF3 silencing markedly inhibited PDGF-BB-induced phenotype switching, proliferation, and migration in VSMCs. Transcriptome sequencing and RNA immunoprecipitation sequencing depicted that ILF3 maintained its stability upon binding to the mRNA of the high-mobility group box 1 protein (HMGB1), thereby exerting an inhibitory effect on the transcription of dual specificity phosphatase 16 (DUSP16) through enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Therefore, the results both in vitro and in vivo indicated that the loss of ILF3 in VSMC ameliorated neointimal hyperplasia by regulating the STAT3/DUSP16 axis through the degradation of HMGB1 mRNA. Our findings revealed that vascular injury activates VSMC ILF3, which in turn promotes intima formation. Consequently, targeting specific VSMC ILF3 may present a potential therapeutic strategy for ameliorating cardiovascular restenosis.

Coptisine inhibits neointimal hyperplasia through attenuating Pak1/Pak2 signaling in vascular smooth muscle cells without retardation of re-endothelialization.

BACKGROUND AND AIMS: Vascular injury-induced endothelium-denudation and profound vascular smooth muscle cells (VSMCs) proliferation and dis-regulated apoptosis lead to post-angioplasty restenosis. Coptisine (CTS), an isoquinoline alkaloid, has multiple beneficial effects on the cardiovascular system. Recent studies identified it selectively inhibits VSMCs proliferation. However, its effects on neointimal hyperplasia, re-endothelialization, and the underlying mechanisms are still unclear. METHODS: Cell viability was assayed by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and cell counting kit-8 (CCK-8). Cell proliferation and apoptosis were measured by flow cytometry and immunofluorescence of Ki67 and TUNEL. Quantitative phosphoproteomics (QPP) was employed to screen CTS-responsive phosphor-sites in the key regulators of cell proliferation and apoptosis. Neointimal hyperplasia was induced by balloon injury of rat left carotid artery (LCA). Adenoviral gene transfer was conducted in both cultured cells and LCA. Re-endothelialization was evaluated by Evan's blue staining of LCA. RESULTS: 1) CTS had strong anti-proliferative and pro-apoptotic effects in cultured rat VSMCs, with the EC50 4∼10-folds lower than that in endothelial cells (ECs). 2) Rats administered with CTS, either locally to LCA's periadventitial space or orally, demonstrated a potently inhibited balloon injury-induced neointimal hyperplasia, but had no delaying effect on re-endothelialization. 3) The QPP results revealed that the phosphorylation levels of Pak1S144/S203, Pak2S20/S197, Erk1T202/Y204, Erk2T185/Y187, and BadS136 were significantly decreased in VSMCs by CTS. 4) Adenoviral expression of phosphomimetic mutants Pak1D144/D203/Pak2D20/D197 enhanced Pak1/2 activities, stimulated the downstream pErk1T202/Y204/pErk2T185/Y187/pErk3S189/pBadS136, attenuated CTS-mediated inhibition of VSMCs proliferation and promotion of apoptosis in vitro, and potentiated neointimal hyperplasia in vivo. 5) Adenoviral expression of phosphoresistant mutants Pak1A144/A203/Pak2A20/A197 inactivated Pak1/2 and totally simulated the inhibitory effects of CTS on platelet-derived growth factor (PDGF)-stimulated VSMCs proliferation and PDGF-inhibited apoptosis in vitro and neointimal hyperplasia in vivo. 6) LCA injury significantly enhanced the endogenous phosphorylation levels of all but pBadS136. CTS markedly attenuated all the enhanced levels. CONCLUSIONS: These results indicate that CTS is a promising medicine for prevention of post-angioplasty restenosis without adverse impact on re-endothelialization. CTS-directed suppression of pPak1S144/S203/pPak2S20/S197 and the subsequent effects on downstream pErk1T202/Y204/pErk2T185/Y187/pErk3S189 and pBadS136 underline its mechanisms of inhibition of VSMCs proliferation and stimulation of apoptosis. Therefore, the phosphor-sites of Pak1S144/S203/Pak2S20/S197 constitute a potential drug-screening target for fighting neointimal hyperplasia restenosis.

Isoxanthohumol reduces neointimal hyperplasia through the apelin/AKT pathway.

The abnormal proliferation, migration, and inflammation of vascular smooth muscle cells (VSMCs) play crucial roles in the development of neointimal hyperplasia and restenosis. Exposure to inflammatory cytokines such as platelet-derived growth factor (PDGF)-BB and tumour necrosis factor-alpha (TNF-α) induces the transformation of contractile VSMCs into abnormal synthetic VSMCs. Isoxanthohumol (IXN) has significant anti-inflammatory, antiproliferative, and antimigratory effects. This study aimed to explore the therapeutic impact and regulatory mechanism of IXN in treating neointimal hyperplasia. The present findings indicate that IXN effectively hinders the abnormal proliferation, migration, and inflammation of VSMCs triggered by PDGF or TNF-α. This inhibition is primarily achieved through the modulation of the apelin/AKT or AKT pathway, respectively. In an in vivo model, IXN effectively reduced neointimal hyperplasia in denuded femoral arteries. These results suggest that IXN holds promise as a potential and innovative therapeutic candidate for the treatment of restenosis.

MTMR7 suppresses the phenotypic switching of vascular smooth muscle cell and vascular intimal hyperplasia after injury via regulating p62/mTORC1-mediated glucose metabolism.

BACKGROUND AND AIMS: Myotubularin-related protein 7 (MTMR7) suppresses proliferation in various cell types and is associated with cardiovascular and cerebrovascular diseases. However, whether MTMR7 regulates vascular smooth muscle cell (VSMC) and vascular intimal hyperplasia remains unclear. We explored the role of MTMR7 in phenotypic switching of VSMC and vascular intimal hyperplasia after injury. METHODS AND RESULTS: MTMR7 expression was significantly downregulated in injured arteries. Compared to wild type (WT) mice, Mtmr7-transgenic (Mtmr7-Tg) mice showed reduced intima/media ratio, decreased percentage of Ki-67-positive cells within neointima, and increased Calponin expression in injured artery. In vitro, upregulating MTMR7 by Len-Mtmr7 transfection inhibited platelet derived growth factor (PDGF)-BB-induced proliferation, migration of VSMC and reversed PDGF-BB-induced decrease in expression of Calponin and SM-MHC. Microarray, single cell sequence, and other bioinformatics analysis revealed that MTMR7 is highly related to glucose metabolism and mammalian target of rapamycin complex 1 (mTORC1). Further experiments confirmed that MTMR7 markedly repressed glycolysis and mTORC1 activity in PDGF-BB-challenged VSMC in vitro. Restoring mTORC1 activity abolished MTMR7-mediated suppression of glycolysis, phenotypic shift in VSMC in vitro and protection against vascular intimal hyperplasia in vivo. Furthermore, upregulating MTMR7 in vitro led to dephosphorylation and dissociation of p62 from mTORC1 in VSMC. External expression of p62 in vitro also abrogated the inhibitory effects of MTMR7 on glycolysis and phenotypic switching in PDGF-BB-stimulated VSMC. CONCLUSIONS: Our study demonstrates that MTMR7 inhibits injury-induced vascular intimal hyperplasia and phenotypic switching of VSMC. Mechanistically, the beneficial effects of MTMR7 are conducted via suppressing p62/mTORC1-mediated glycolysis.

Metabolic reprogramming of immune cells by mitochondrial division inhibitor-1 to prevent post-vascular injury neointimal hyperplasia.

BACKGROUND AND AIMS: New treatments are needed to prevent neointimal hyperplasia that contributes to post-angioplasty and stent restenosis in patients with coronary artery disease (CAD) and peripheral arterial disease (PAD). We investigated whether modulating mitochondrial function using mitochondrial division inhibitor-1 (Mdivi-1) could reduce post-vascular injury neointimal hyperplasia by metabolic reprogramming of macrophages from a pro-inflammatory to anti-inflammatory phenotype. METHODS AND RESULTS: In vivo Mdivi-1 treatment of Apoe-/- mice fed a high-fat diet and subjected to carotid-wire injury decreased neointimal hyperplasia by 68%, reduced numbers of plaque vascular smooth muscle cells and pro-inflammatory M1-like macrophages, and decreased plaque inflammation, endothelial activation, and apoptosis, when compared to control. Mdivi-1 treatment of human THP-1 macrophages shifted polarization from a pro-inflammatory M1-like to an anti-inflammatory M2-like phenotype, reduced monocyte chemotaxis and migration to CCL2 and macrophage colony stimulating factor (M-CSF) and decreased secretion of pro-inflammatory mediators. Finally, treatment of pro-inflammatory M1-type-macrophages with Mdivi-1 metabolically reprogrammed them to an anti-inflammatory M2-like phenotype by inhibiting oxidative phosphorylation and attenuating the increase in succinate levels and correcting the decreased levels of arginine and citrulline. CONCLUSIONS: We report that treatment with Mdivi-1 inhibits post-vascular injury neointimal hyperplasia by metabolic reprogramming macrophages towards an anti-inflammatory phenotype thereby highlighting the therapeutic potential of Mdivi-1 for preventing neointimal hyperplasia and restenosis following angioplasty and stenting in CAD and PAD patients.

Transfer RNA-derived small RNA tRF-Glu-CTC attenuates neointimal formation via inhibition of fibromodulin.

Neointimal hyperplasia is a pathological vascular remodeling caused by abnormal proliferation and migration of subintimal vascular smooth muscle cells (VSMCs) following intimal injury. There is increasing evidence that tRNA-derived small RNA (tsRNA) plays an important role in vascular remodeling. The purpose of this study is to search for tsRNAs signature of neointima formation and to explore their potential functions. The balloon injury model of rat common carotid artery was replicated to induce intimal hyperplasia, and the differentially expressed tsRNAs (DE-tsRNAs) in arteries with intimal hyperplasia were screened by small RNA sequencing and tsRNA library. A total of 24 DE-tsRNAs were found in the vessels with intimal hyperplasia by small RNA sequencing. In vitro, tRF-Glu-CTC inhibited the expression of fibromodulin (FMOD) in VSMCs, which is a negative modulator of TGF-ß1 activity. tRF-Glu-CTC also increased VSMC proliferation and migration. In vivo experiments showed that inhibition of tRF-Glu-CTC expression after balloon injury of rat carotid artery can reduce the neointimal area. In conclusion, tRF-Glu-CTC expression is increased after vascular injury and inhibits FMOD expression in VSMCs, which influences neointima formation. On the other hand, reducing the expression of tRF-Glu-CTC after vascular injury may be a potential approach to prevent vascular stenosis.

Vascular injury activates the ELK1/SND1/SRF pathway to promote vascular smooth muscle cell proliferative phenotype and neointimal hyperplasia.

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation is the leading cause of vascular stenosis or restenosis. Therefore, investigating the molecular mechanisms and pivotal regulators of the proliferative VSMC phenotype is imperative for precisely preventing neointimal hyperplasia in vascular disease. METHODS: Wire-induced vascular injury and aortic culture models were used to detect the expression of staphylococcal nuclease domain-containing protein 1 (SND1). SMC-specific Snd1 knockout mice were used to assess the potential roles of SND1 after vascular injury. Primary VSMCs were cultured to evaluate SND1 function on VSMC phenotype switching, as well as to investigate the mechanism by which SND1 regulates the VSMC proliferative phenotype. RESULTS: Phenotype-switched proliferative VSMCs exhibited higher SND1 protein expression compared to the differentiated VSMCs. This result was replicated in primary VSMCs treated with platelet-derived growth factor (PDGF). In the injury model, specific knockout of Snd1 in mouse VSMCs reduced neointimal hyperplasia. We then revealed that ETS transcription factor ELK1 (ELK1) exhibited upregulation and activation in proliferative VSMCs, and acted as a novel transcription factor to induce the gene transcriptional activation of Snd1. Subsequently, the upregulated SND1 is associated with serum response factor (SRF) by competing with myocardin (MYOCD). As a co-activator of SRF, SND1 recruited the lysine acetyltransferase 2B (KAT2B) to the promoter regions leading to the histone acetylation, consequently promoted SRF to recognize the specific CArG motif, and enhanced the proliferation- and migration-related gene transcriptional activation. CONCLUSIONS: The present study identifies ELK1/SND1/SRF as a novel pathway in promoting the proliferative VSMC phenotype and neointimal hyperplasia in vascular injury, predisposing the vessels to pathological remodeling. This provides a potential therapeutic target for vascular stenosis.

Zic family member 3 attenuates oxidative stress-induced vascular smooth muscle cell apoptosis in patients with chronic kidney disease.

Neointimal hyperplasia is reportedly essential for arteriovenous fistulas (AVF) in patients undergoing hemodialysis. Oxidative stress is vital in the progression of uremic venous intimal hyperplasia. Studies have suggested that zinc ions obstruct vascular calcification in patients with chronic kidney disease (CKD). Recent studies have shown that the zinc finger protein, Zic family member 3 (ZIC3), is crucial for the earliest cardiovascular progenitors. ZIC3 mutations are associated with congenital heart disease. However, the mechanism of action of ZIC3 in vascular intimal hyperplasia in CKD remains unelucidated. Venous specimens were collected during primary AVF surgery and traumatic amputation, and serum samples were collected from patients with CKD and healthy controls. Mouse vascular smooth muscle cells (VSMCs) were treated with hydrogen peroxide (H2O2) to clarify the role of ZIC3 in CKD. ZIC3 expression was reduced in the veins of patients with uremia and the serum of those with CKD. Zic3 and Bcl2 levels were significantly decreased in mouse VSMCs treated with H2O2·H2O2 inhibited mouse VSMC activity, upregulated Bax, and cleaved caspase 3 expression. Following Zic3 overexpression, Bcl2 expression level and cell viability were elevated, whereas Bax and cleaved caspase 3 expression levels were downregulated. In contrast, Zic3 knockdown yielded the opposite results. Therefore, ZIC3 could be a new therapeutic target in venous neointimal hyperplasia of CKD.

Myeloid-derived growth factor suppresses VSMC dedifferentiation and attenuates postinjury neointimal formation in rats by activating S1PR2 and its downstream signaling.

Restenosis after angioplasty is caused usually by neointima formation characterized by aberrant vascular smooth muscle cell (VSMC) dedifferentiation. Myeloid-derived growth factor (MYDGF), secreted from bone marrow-derived monocytes and macrophages, has been found to have cardioprotective effects. In this study we investigated the effect of MYDGF to postinjury neointimal formation and the underlying mechanisms. Rat carotid arteries balloon-injured model was established. We found that plasma MYDGF content and the level of MYDGF in injured arteries were significantly decreased after balloon injury. Local application of exogenous MYDGF (50 µg/mL) around the injured vessel during balloon injury markedly ameliorated the development of neointimal formation evidenced by relieving the narrow endovascular diameter, improving hemodynamics, and reducing collagen deposition. In addition, local application of MYDGF inhibited VSMC dedifferentiation, which was proved by reversing the elevated levels of osteopontin (OPN) protein and decreased levels of α-smooth muscle actin (α-SMA) in the left carotid arteries. We showed that PDGF-BB (30 ng/mL) stimulated VSMC proliferation, migration and dedifferentiation in vitro; pretreatment with MYDGF (50-200 ng/mL) concentration-dependently eliminated PDGF-BB-induced cell proliferation, migration and dedifferentiation. Molecular docking revealed that MYDGF had the potential to bind with sphingosine-1-phosphate receptor 2 (S1PR2), which was confirmed by SPR assay and Co-IP analysis. Pretreatment with CCG-1423 (Rho signaling inhibitor), JTE-013 (S1PR2 antagonist) or Ripasudil (ROCK inhibitor) circumvented the inhibitory effects of MYDGF on VSMC phenotypic switching through inhibiting S1PR2 or its downstream RhoA-actin monomers (G-actin) /actin filaments (F-actin)-MRTF-A signaling. In summary, this study proves that MYDGF relieves neointimal formation of carotid arteries in response to balloon injury in rats, and suppresses VSMC dedifferentiation induced by PDGF-BB via S1PR2-RhoA-G/F-actin-MRTF-A signaling pathway. In addition, our results provide evidence for cross talk between bone marrow and vasculature.

Effect of F11 Receptor/Junctional Adhesion Molecule-A-derived Peptide on Neointimal Hyperplasia in a Murine Model.

PURPOSE: To determine whether inhibition of the F11 receptor/JAM-A (F11R) using F11R-specific antagonist peptide 4D results in inhibition of smooth muscle cell (SMC) proliferation and migration in vivo, known as neointimal hyperplasia (NIH), using a mouse focal carotid artery stenosis model (FCASM). MATERIALS AND METHODS: The mouse FCASM was chosen to test the hypothesis because the dominant cell type at the site of stenosis is SMC, similar to that in vascular access stenosis. Fourteen C57BL/6 mice underwent left carotid artery (LCA) partial ligation to induce stenosis, followed by daily injection of peptide 4D in 7 mice and saline in the remaining 7 mice, and these mice were observed for 21 days and then euthanized. Bilateral carotid arteries were excised for histologic analysis of the intima and media areas. RESULTS: The mean intimal area was significantly larger in control mice compared with peptide 4D-treated mice (0.031 mm2 [SD ± 0.024] vs 0.0082 mm2 [SD ± 0.0103]; P = .011). The mean intima-to-intima + media area ratio was significantly larger in control mice compared with peptide 4D-treated mice (0.27 [SD ± 0.13] vs 0.089 [SD ± 0.081]; P = .0079). NIH was not observed in the right carotid arteries in both groups. CONCLUSIONS: Peptide 4D, an F11R antagonist, significantly inhibited NIH in C57BL/6 mice in a FCASM.

Severe arterial injury heals with a complex clonal structure involving a large fraction of surviving smooth muscle cells.

BACKGROUND AND AIMS: Smooth muscle cell (SMC) lineage cells in atherosclerosis and flow cessation-induced neointima are oligoclonal, being recruited from a tiny fraction of medial SMCs that modulate and proliferate. The present study aimed to investigate the clonal structure of SMC lineage cells healing more severe arterial injury. METHODS: Arterial injury (wire, stretch, and partial ligation) was inflicted on the right carotid artery in mice with homozygous, SMC-restricted, stochastically recombining reporter transgenes that produced mosaic expression of 10 distinguishable fluorescent phenotypes for clonal tracking. Healed arteries and contra-lateral controls were analyzed after 3 weeks. Additional analysis of cell death and proliferation after injury was performed in wildtype mice. RESULTS: The total number of SMC lineage cells in healed arteries was comparable to normal arteries but comprised significantly fewer fluorescent phenotypes. The population had a complex, intermixed, clonal structure. By statistical analysis of expected versus observed fractions of fluorescent phenotypes and visual inspection of coherent groups of same-colored cells, we concluded that >98% of SMC lineage cells in healed arteries belonged to a detectable clone, indicating that nearly all surviving SMCs after severe injury at some point undergo proliferation. This was consistent with serial observations in the first week after injury, which showed severe loss of medial cells followed by widespread proliferation. CONCLUSIONS: After severe arterial injury, many surviving SMCs proliferate to repair the media and form a neointima. This indicates that the fraction of medial SMCs that are mobilized to repair arteries increases with the level of injury.

RhoGDI3 at the trans-Golgi network participates in NLRP3 inflammasome activation, VSMC phenotypic modulation, and neointima formation.

BACKGROUND AND AIMS: The pathological roles and mechanisms of Rho-specific guanine nucleotide dissociation inhibitor 3 (RhoGDI3) in vascular smooth muscle cell (VSMC) phenotypic modulation and neointima formation are currently unknown. This study aimed to investigate how RhoGDI3 regulates the Nod-like receptor protein 3 (NLRP3) inflammasome in platelet-derived growth factor-BB (PDGF-BB)-induced neointima formation. METHODS: For in vitro assays, human aortic VSMCs (HA-VSMCs) were transfected with pcDNA3.1-GDI3 and RhoGDI3 siRNA to overexpress and knockdown RhoGDI3, respectively. HA-VSMCs were also treated with an NLRP3 inhibitor (CY-09) or agonist (NSS). Protein transcription and expression, cell proliferation and migration, Golgi morphology, and protein binding and colocalization were measured. For the in vivo assays, balloon injury (BI) rats were injected with recombinant adenovirus carrying RhoGDI3 shRNA. Carotid arterial morphology, protein expression and colocalization, and activation of the NLRP3 inflammasome were measured. RESULTS: PDGF-BB treatment induced transcription and expression of RhoGDI3 through PDGF receptor αß (PDGFRαß) rather than PDGFRαα or PDGFRßß in HA-VSMCs. RhoGDI3 suppression blocked PDGF-BB-induced VSMC phenotypic transformation. In contrast, RhoGDI3 overexpression further promoted PDGF-BB-induced VSMC dedifferentiation. The in vivo results also confirmed that RhoGDI3 expressed in VSMCs participated in neointima formation and muscle fiber and collagen deposition caused by balloon injury. In addition, PDGF-BB increased binding of RhoGDI3 to NLRP3 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) at the trans-Golgi membrane, which depended on the normal Golgi network. However, recruitment of NLRP3 and ASC to the trans-Golgi network after PDGF-BB treatment was independent of RhoGDI3. Moreover, RhoGDI3 knockdown significantly inhibited ASC expression and NLRP3 inflammasome assembly and activation and reduced NLRP3 protein stability in PDGF-BB-treated HA-VSMCs. Inhibiting NLRP3 effectively prevented PDGF-BB-induced VSMC phenotypic modulation, and an NLRP3 agonist reversed the decline in VSMC phenotypic transformation caused by RhoGDI3 knockdown. Furthermore, RhoGDI3 suppression reduced the protein levels and assembly of NLRP3 and ASC, and the activation of the NLRP3 inflammasome in VSMCs in a rat balloon injury model. CONCLUSIONS: The results of this study reveal a novel mechanism through which RhoGDI3 regulates VSMC phenotypic modulation and neointima formation by activating the NLRP3 inflammasome.

P4HA2-induced prolyl hydroxylation of YAP1 restricts vascular smooth muscle cell proliferation and neointima formation.

Vascular smooth muscle cell (VSMC) proliferation and neointima formation play significant roles in atherosclerosis development and restenosis following percutaneous coronary intervention. Our team previously discovered that TEA domain transcription factor 1 (TEAD1) promotes vascular smooth muscle differentiation, which is necessary for vascular development. Conversely, aberrant YAP1 activation upregulates the platelet-derived growth factor receptor beta to encourage VSMC proliferation and neointima formation. In this study, we aimed to investigate the molecular mechanisms of YAP1/TEAD signaling during neointima formation. Our research focused on the prolyl 4-hydroxylase alpha 2 (P4HA2) and its downstream target, Yes-associated protein 1 (YAP1), in regulating VSMC differentiation and neointima formation. Our results indicated that P4HA2 reduction leads to VSMC dedifferentiation and promotes neointima formation after injury. Furthermore, we found that P4HA2-induced prolyl hydroxylation of YAP1 restricts its transcriptional activity, which is essential to maintaining VSMC differentiation. These findings suggest that targeting P4HA2-mediated prolyl hydroxylation of YAP1 may be a promising therapeutic approach to prevent injury-induced neointima formation in cardiovascular disease.

Ketogenic diet inhibits neointimal hyperplasia by suppressing oxidative stress and inflammation.

OBJECTIVE: Neointimal hyperplasia is the primary mechanism underlying atherosclerosis and restenosis after percutaneous coronary intervention. Ketogenic diet (KD) exerts beneficial effects in various diseases, but whether it could serve as non-drug therapy for neointimal hyperplasia remains unknown. This study aimed to investigate the effect of KD on neointimal hyperplasia and the potential mechanisms. METHODS AND RESULTS: Carotid artery balloon-injury model was employed in adult Sprague-Dawley rats to induce neointimal hyperplasia. Then, animals were subjected to either standard rodent chow or KD. For in-vitro experiment, impacts of ß-hydroxybutyrate (ß-HB), the main mediator of KD effects, on platelet-derived growth factor BB (PDGF-BB) induced vascular smooth muscle cell (VSMC) migration and proliferation were determined. Balloon injury induced event intimal hyperplasia and upregulation of protein expression of proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA), and these changes were significantly ameliorated by KD. In addition, ß-HB could markedly inhibit PDGF-BB induced VMSC migration and proliferation, as well as inhibiting expressions of PCNA and α-SMC. Furthermore, KD inhibited balloon-injury induced oxidative stress in carotid artery, indicated by reduced ROS level, malondialdehyde (MDA) and myeloperoxidase (MPO) activities, and increased superoxide dismutase (SOD) activity. We also found balloon-injury induced inflammation in carotid artery was suppressed by KD, indicated by decreased expressions of proinflammatory cytokines IL-1ß and TNF-α, and increased expression of anti-inflammatory cytokine IL-10. CONCLUSION: KD attenuates neointimal hyperplasia through suppressing oxidative stress and inflammation to inhibit VSMC proliferation and migration. KD may represent a promising non-drug therapy for neointimal hyperplasia associated diseases.

Sox10 escalates vascular inflammation by mediating vascular smooth muscle cell transdifferentiation and pyroptosis in neointimal hyperplasia.

Vascular smooth muscle cells (VSMCs) can transdifferentiate into macrophage-like cells in the context of sustained inflammatory injury, which drives vascular hyperplasia and atherosclerotic complications. Using single-cell RNA sequencing, we identify that macrophage-like VSMCs are the key cell population in mouse neointimal hyperplasia. Sex-determining region Y (SRY)-related HMG-box gene 10 (Sox10) upregulation is associated with macrophage-like VSMC accumulation and pyroptosis in vitro and in the neointimal hyperplasia of mice. Tumor necrosis factor α (TNF-α)-induced Sox10 lactylation in a phosphorylation-dependent manner by PI3K/AKT signaling drives transcriptional programs of VSMC transdifferentiation, contributing to pyroptosis. The regulator of G protein signaling 5 (RGS5) interacts with AKT and blocks PI3K/AKT signaling and Sox10 phosphorylation at S24. Sox10 silencing mitigates vascular inflammation and forestalls neointimal hyperplasia in RGS5 knockout mice. Collectively, this study shows that Sox10 is a regulator of vascular inflammation and a potential control point in inflammation-related vascular disease.

BRD4770 inhibits vascular smooth muscle cell proliferation via SUV39H2, but not EHMT2 to protect against neointima formation.

The behavior of vascular smooth muscle cells (VSMCs) contributes to the formation of neointima. We previously found that EHMT2 suppressed autophagy activation in VSMCs. BRD4770, an inhibitor of EHMT2/G9a, plays a critical role in several kinds of cancers. However, whether and how BRD4770 regulates the behavior of VSMCs remain unknown. In this study, we evaluate the cellular effect of BRD4770 on VSMCs by series of experiments in vivo and ex vivo. We demonstrated that BRD4770 inhibited VSMCs' growth by blockage in G2/M phase in VSMCs. Moreover, our results demonstrated that the inhibition of proliferation was independent on autophagy or EHMT2 suppression which we previous reported. Mechanistically, BRD4770 exhibited an off-target effect from EHMT2 and our further study reveal that the proliferation inhibitory effect by BRD4770 was associated with suppressing on SUV39H2/KTM1B. In vivo, BRD4770 was also verified to rescue VIH. Thus, BRD4770 function as a crucial negative regulator of VSMC proliferation via SUV39H2 and G2/M cell cycle arrest and BRD4770 could be a molecule for the therapy of vascular restenosis.