Confounding Effects of Tamoxifen: Cautionary and Practical Considerations for the Use of Tamoxifen-Inducible Mouse Models in Atherosclerosis Research-Brief Report.

BACKGROUND: In recent years, fate-mapping lineage studies in mouse models have led to major advances in vascular biology by allowing investigators to track specific cell populations in vivo. One of the most frequently used lineage tracing approaches involves tamoxifen-inducible CreERT-LoxP systems. However, tamoxifen treatment can also promote effects independent of Cre recombinase activation, many of which have not been fully explored. METHODS: To elucidate off-target effects of tamoxifen, male and female mice were either unmanipulated or injected with tamoxifen or corn oil. All mice received PCSK9 (proprotein convertase subtilisin/kexin type 9)-AAV (adeno-associated virus) injections and a modified Western diet to induce hypercholesterolemia. After 2 weeks, serum cholesterol and liver morphology were assessed. To determine the duration of any tamoxifen effects in long-term atherosclerosis experiments, mice received either 12 days of tamoxifen at baseline or 12 days plus 2 sets of 5-day tamoxifen boosters; all mice received PCSK9-AAV injections and a modified Western diet to induce hypercholesterolemia. After 24 weeks, serum cholesterol and aortic sinus plaque burden were measured. RESULTS: After 2 weeks of atherogenic treatment, mice injected with tamoxifen demonstrated significantly reduced serum cholesterol levels compared with uninjected- or corn oil-treated mice. However, there were no differences in PCSK9-mediated knockdown of LDL (low-density lipoprotein) receptors between the groups. Additionally, tamoxifen-treated mice exhibited significantly increased hepatic lipid accumulation compared with the other groups. Finally, the effects of tamoxifen remained for at least 8 weeks after completion of injections, with mice demonstrating persistent decreased serum cholesterol and impaired atherosclerotic plaque formation. CONCLUSIONS: In this study, we establish that tamoxifen administration results in decreased serum cholesterol, decreased plaque formation, and increased hepatic lipid accumulation. These alterations represent significant confounding variables in atherosclerosis research, and we urge future investigators to take these findings into consideration when planning and executing their own atherosclerosis experiments.

Genes That Escape X Chromosome Inactivation Modulate Sex Differences in Valve Myofibroblasts.

BACKGROUND: Aortic valve stenosis is a sexually dimorphic disease, with women often presenting with sustained fibrosis and men with more extensive calcification. However, the intracellular molecular mechanisms that drive these clinically important sex differences remain underexplored. METHODS: Hydrogel biomaterials were designed to recapitulate key aspects of the valve tissue microenvironment and to serve as a culture platform for sex-specific valvular interstitial cells (VICs; precursors to profibrotic myofibroblasts). The hydrogel culture system was used to interrogate intracellular pathways involved in sex-dependent VIC-to-myofibroblast activation and deactivation. RNA sequencing was used to define pathways involved in driving sex-dependent activation. Interventions with small molecule inhibitors and siRNA transfections were performed to provide mechanistic insight into sex-specific cellular responses to microenvironmental cues, including matrix stiffness and exogenously delivered biochemical factors. RESULTS: In both healthy porcine and human aortic valves, female leaflets had higher baseline activation of the myofibroblast marker α-smooth muscle actin compared with male leaflets. When isolated and cultured, female porcine and human VICs had higher levels of basal α-smooth muscle actin stress fibers that further increased in response to the hydrogel matrix stiffness, both of which were higher than in male VICs. A transcriptomic analysis of male and female porcine VICs revealed Rho-associated protein kinase signaling as a potential driver of this sex-dependent myofibroblast activation. Furthermore, we found that genes that escape X-chromosome inactivation such as BMX and STS (encoding for Bmx nonreceptor tyrosine kinase and steroid sulfatase, respectively) partially regulate the elevated female myofibroblast activation through Rho-associated protein kinase signaling. This finding was confirmed by treating male and female VICs with endothelin-1 and plasminogen activator inhibitor-1, factors that are secreted by endothelial cells and known to drive myofibroblast activation through Rho-associated protein kinase signaling. CONCLUSIONS: Together, in vivo and in vitro results confirm sex dependencies in myofibroblast activation pathways and implicate genes that escape X-chromosome inactivation in regulating sex differences in myofibroblast activation and subsequent aortic valve stenosis progression. Our results underscore the importance of considering sex as a biological variable to understand the molecular mechanisms of aortic valve stenosis and to help guide sex-based precision therapies.

PTEN (Phosphatase and Tensin Homolog) Protects Against Ang II (Angiotensin II)-Induced Pathological Vascular Fibrosis and Remodeling-Brief Report.

OBJECTIVE: Pathological vascular remodeling and excessive perivascular fibrosis are major contributors to reduced vessel compliance that exacerbates cardiovascular diseases, for instance, promoting clinically relevant myocardial remodeling. Inflammation plays a significant role in both pathological vascular remodeling and fibrosis. We previously demonstrated that smooth muscle cell-specific PTEN depletion promotes significant vascular fibrosis and accumulation of inflammatory cells. In the current study, we aimed to determine the beneficial role of systemic PTEN elevation on Ang II (angiotensin II)-induced vascular fibrosis and remodeling. Approach and Results: Transgenic mice carrying additional copies of the wild-type Pten gene (super PTEN [sPTEN]) and WT littermates were subjected to Ang II or saline infusion for 14 or 28 days. Compared with WT, Ang II-induced vascular fibrosis was significantly blunted in sPTEN mice, as shown by histochemical stainings and label-free second harmonic generation imaging. The protection against Ang II was recapitulated in sPTEN mice bearing WT bone marrow but not in WT mice reconstituted with sPTEN bone marrow. Ang II-induced elevation of profibrotic and proinflammatory gene expression observed in WT mice was blocked in aortic tissue of sPTEN mice. Immunofluorescent staining and flow cytometry both indicated that perivascular infiltration of T cells and macrophages was significantly inhibited in sPTEN mice. In vitro induction of PTEN expression suppressed Ang II-induced Ccl2 expression in vascular smooth muscle cells. CONCLUSIONS: Systemic PTEN elevation mediates protection against Ang II-induced vascular inflammation and fibrosis predominantly through effects in resident vascular cells. Our data highly support that pharmacological upregulation of PTEN could be a novel and viable approach for the treatment of pathological vascular fibrosis.

High Throughput Screen Identifies the DNMT1 (DNA Methyltransferase-1) Inhibitor, 5-Azacytidine, as a Potent Inducer of PTEN (Phosphatase and Tensin Homolog): Central Role for PTEN in 5-Azacytidine Protection Against Pathological Vascular Remodeling.

OBJECTIVE: Our recent work demonstrates that PTEN (phosphatase and tensin homolog) is an important regulator of smooth muscle cell (SMC) phenotype. SMC-specific PTEN deletion promotes spontaneous vascular remodeling and PTEN loss correlates with increased atherosclerotic lesion severity in human coronary arteries. In mice, PTEN overexpression reduces plaque area and preserves SMC contractile protein expression in atherosclerosis and blunts Ang II (angiotensin II)-induced pathological vascular remodeling, suggesting that pharmacological PTEN upregulation could be a novel therapeutic approach to treat vascular disease. Approach and Results: To identify novel PTEN activators, we conducted a high-throughput screen using a fluorescence based PTEN promoter-reporter assay. After screening ≈3400 compounds, 11 hit compounds were chosen based on level of activity and mechanism of action. Following in vitro confirmation, we focused on 5-azacytidine, a DNMT1 (DNA methyltransferase-1) inhibitor, for further analysis. In addition to PTEN upregulation, 5-azacytidine treatment increased expression of genes associated with a differentiated SMC phenotype. 5-Azacytidine treatment also maintained contractile gene expression and reduced inflammatory cytokine expression after PDGF (platelet-derived growth factor) stimulation, suggesting 5-azacytidine blocks PDGF-induced SMC de-differentiation. However, these protective effects were lost in PTEN-deficient SMCs. These findings were confirmed in vivo using carotid ligation in SMC-specific PTEN knockout mice treated with 5-azacytidine. In wild type controls, 5-azacytidine reduced neointimal formation and inflammation while maintaining contractile protein expression. In contrast, 5-azacytidine was ineffective in PTEN knockout mice, indicating that the protective effects of 5-azacytidine are mediated through SMC PTEN upregulation. CONCLUSIONS: Our data indicates 5-azacytidine upregulates PTEN expression in SMCs, promoting maintenance of SMC differentiation and reducing pathological vascular remodeling in a PTEN-dependent manner.

KLF4 in smooth muscle cell-derived progenitor cells is essential for angiotensin II-induced cardiac inflammation and fibrosis.

Cardiac fibrosis is defined by the excessive accumulation of extracellular matrix (ECM) material resulting in cardiac tissue scarring and dysfunction. While it is commonly accepted that myofibroblasts are the major contributors to ECM deposition in cardiac fibrosis, their origin remains debated. By combining lineage tracing and RNA sequencing, our group made the paradigm-shifting discovery that a subpopulation of resident vascular stem cells residing within the aortic, carotid artery, and femoral aartery adventitia (termed AdvSca1-SM cells) originate from mature vascular smooth muscle cells (SMCs) through an in situ reprogramming process. SMC-to-AdvSca1-SM reprogramming and AdvSca1-SM cell maintenance is dependent on induction and activity of the transcription factor, KLF4. However, the molecular mechanism whereby KLF4 regulates AdvSca1-SM phenotype remains unclear. In the current study, leveraging a highly specific AdvSca1-SM cell reporter system, single-cell RNA-sequencing (scRNA-seq), and spatial transcriptomic approaches, we demonstrate the profibrotic differentiation trajectory of coronary artery-associated AdvSca1-SM cells in the setting of Angiotensin II (AngII)-induced cardiac fibrosis. Differentiation was characterized by loss of stemness-related genes, including Klf4 , but gain of expression of a profibrotic phenotype. Importantly, these changes were recapitulated in human cardiac hypertrophic tissue, supporting the translational significance of profibrotic transition of AdvSca1-SM-like cells in human cardiomyopathy. Surprisingly and paradoxically, AdvSca1-SM-specific genetic knockout of Klf4 prior to AngII treatment protected against cardiac inflammation and fibrosis, indicating that Klf4 is essential for the profibrotic response of AdvSca1-SM cells. Overall, our data reveal the contribution of AdvSca1-SM cells to myofibroblasts in the setting of AngII-induced cardiac fibrosis. KLF4 not only maintains the stemness of AdvSca1-SM cells, but also orchestrates their response to profibrotic stimuli, and may serve as a therapeutic target in cardiac fibrosis.

Smooth muscle-derived adventitial progenitor cells promote key cell type transitions controlling plaque stability in atherosclerosis in a Klf4-dependent manner.

We previously established that vascular smooth muscle-derived adventitial progenitor cells (AdvSca1-SM) preferentially differentiate into myofibroblasts and contribute to fibrosis in response to acute vascular injury. However, the role of these progenitor cells in chronic atherosclerosis has not been defined. Using an AdvSca1-SM lineage tracing model, scRNA-Seq, flow cytometry, and histological approaches, we confirmed that AdvSca1-SM cells localize throughout the vessel wall and atherosclerotic plaques, where they primarily differentiate into fibroblasts, SMCs, or remain in a stem-like state. Klf4 knockout specifically in AdvSca1-SM cells induced transition to a more collagen-enriched myofibroblast phenotype compared to WT mice. Additionally, Klf4 depletion drastically modified the phenotypes of non-AdvSca1-SM-derived cells, resulting in more contractile SMCs and atheroprotective macrophages. Functionally, overall plaque burden was not altered with Klf4 depletion, but multiple indices of plaque vulnerability were reduced. Collectively, these data support that modulating the AdvSca1-SM population confers increased protection from the development of unstable atherosclerotic plaques.

Redistribution of the chromatin remodeler Brg1 directs smooth muscle-derived adventitial progenitor-to-myofibroblast differentiation and vascular fibrosis.

Vascular smooth muscle-derived Sca1+ adventitial progenitor (AdvSca1-SM) cells are tissue-resident, multipotent stem cells that contribute to progression of vascular remodeling and fibrosis. Upon acute vascular injury, AdvSca1-SM cells differentiate into myofibroblasts and are embedded in perivascular collagen and the extracellular matrix. While the phenotypic properties of AdvSca1-SM-derived myofibroblasts have been defined, the underlying epigenetic regulators driving the AdvSca1-SM-to-myofibroblast transition are unclear. We show that the chromatin remodeler Smarca4/Brg1 facilitates AdvSca1-SM myofibroblast differentiation. Brg1 mRNA and protein were upregulated in AdvSca1-SM cells after acute vascular injury, and pharmacological inhibition of Brg1 by the small molecule PFI-3 attenuated perivascular fibrosis and adventitial expansion. TGF-ß1 stimulation of AdvSca1-SM cells in vitro reduced expression of stemness genes while inducing expression of myofibroblast genes that was associated with enhanced contractility; PFI blocked TGF-ß1-induced phenotypic transition. Similarly, genetic knockdown of Brg1 in vivo reduced adventitial remodeling and fibrosis and reversed AdvSca1-SM-to-myofibroblast transition in vitro. Mechanistically, TGF-ß1 promoted redistribution of Brg1 from distal intergenic sites of stemness genes and recruitment to promoter regions of myofibroblast-related genes, which was blocked by PFI-3. These data provide insight into epigenetic regulation of resident vascular progenitor cell differentiation and support that manipulating the AdvSca1-SM phenotype will provide antifibrotic clinical benefits.

Smooth muscle-derived adventitial progenitor cells direct atherosclerotic plaque composition complexity in a Klf4-dependent manner.

We previously established that vascular smooth muscle-derived adventitial progenitor cells (AdvSca1-SM) preferentially differentiate into myofibroblasts and contribute to fibrosis in response to acute vascular injury. However, the role of these progenitor cells in chronic atherosclerosis has not been defined. Using an AdvSca1-SM cell lineage tracing model, scRNA-Seq, flow cytometry, and histological approaches, we confirmed that AdvSca1-SM-derived cells localized throughout the vessel wall and atherosclerotic plaques, where they primarily differentiated into fibroblasts, smooth muscle cells (SMC), or remained in a stem-like state. Krüppel-like factor 4 (Klf4) knockout specifically in AdvSca1-SM cells induced transition to a more collagen-enriched fibroblast phenotype compared with WT mice. Additionally, Klf4 deletion drastically modified the phenotypes of non-AdvSca1-SM-derived cells, resulting in more contractile SMC and atheroprotective macrophages. Functionally, overall plaque burden was not altered with Klf4 deletion, but multiple indices of plaque composition complexity, including necrotic core area, macrophage accumulation, and fibrous cap thickness, were reduced. Collectively, these data support that modulation of AdvSca1-SM cells through KLF4 depletion confers increased protection from the development of potentially unstable atherosclerotic plaques.

Heterogeneous subpopulations of adventitial progenitor cells regulate vascular homeostasis and pathological vascular remodelling.

Cardiovascular diseases are characterized by chronic vascular dysfunction and provoke pathological remodelling events, such as neointima formation, atherosclerotic lesion development, and adventitial fibrosis. While lineage-tracing studies have shown that phenotypically modulated smooth muscle cells (SMCs) are the major cellular component of neointimal lesions, the cellular origins and microenvironmental signalling mechanisms that underlie remodelling along the adventitial vascular layer are not fully understood. However, a growing body of evidence supports a unique population of adventitial lineage-restricted progenitor cells expressing the stem cell marker, stem cell antigen-1 (Sca1; AdvSca1 cells) as important effectors of adventitial remodelling and suggests that they are at least partially responsible for subsequent pathological changes that occur in the media and intima. AdvSca1 cells are being studied in murine models of atherosclerosis, perivascular fibrosis, and neointima formation in response to acute vascular injury. Depending on the experimental conditions, AdvSca1 cells exhibit the capacity to differentiate into SMCs, endothelial cells, chondrocytes, adipocytes, and pro-remodelling cells, such as myofibroblasts and macrophages. These data indicate that AdvSca1 cells may be a targetable cell population to influence the outcomes of pathologic vascular remodelling. Important questions remain regarding the origins of AdvSca1 cells and the essential signalling mechanisms and microenvironmental factors that regulate both maintenance of their stem-like, progenitor phenotype and their differentiation into lineage-specified cell types. Adding complexity to the story, recent data indicate that the collective population of adventitial progenitor cells is likely composed of several smaller, lineage-restricted subpopulations, which are not fully defined by their transcriptomic profile and differentiation capabilities. The aim of this review is to outline the heterogeneity of Sca1+ adventitial progenitor cells, summarize their role in vascular homeostasis and remodelling, and comment on their translational relevance in humans.

Urinary matrix metalloproteinase activities: biomarkers for plaque angiogenesis and nephropathy in diabetes.

Diabetic complications of nephropathy and accelerated atherosclerosis are associated with vascular remodeling and dysregulated angiogenesis. Matrix metalloproteinases (MMP) modify extracellular matrix during vascular remodeling and are excreted in urine of patients with vascular malformation or tumor angiogenesis. We hypothesized that urinary MMP activities would be sensitive biomarkers for vascular remodeling in diabetic complications. Activities of MMP-2, MMP-9, and its complex with neutrophil gelatinase-associated lipocalin (NGAL/MMP-9) were measured by substrate gel zymography in urine from nondiabetic (ND) and type 1 diabetic (T1D) rodents that were susceptible to both T1D-induced plaque angiogenesis and nephropathy, or nephropathy alone. Additionally, these urine activities were measured in ND and T1D adolescents. Urinary MMP-9, MMP-2, and NGAL/MMP-9 activities were increased and more prevalent in T1D compared with ND controls. Urinary MMP-2 activity was detected in mice with T1D-induced plaque neovascularization. In nephropathy models, urinary NGAL/MMP-9 and MMP-9 activities appeared before onset of albuminuria, whereas MMP-2 was absent or delayed. Finally, urinary MMP activities were increased in adolescents with early stages of T1D. Urinary MMP activities may be sensitive, noninvasive, and clinically useful biomarkers for predicting vascular remodeling in diabetic renal and vascular complications.

Matrix-Degrading Enzyme Expression and Aortic Fibrosis During Continuous-Flow Left Ventricular Mechanical Support.

BACKGROUND: The effects of nonphysiological flow generated by continuous-flow (CF) left ventricular assist devices (LVADs) on the aorta remain poorly understood. OBJECTIVES: The authors sought to quantify indexes of fibrosis and determine the molecular signature of post-CF-LVAD vascular remodeling. METHODS: Paired aortic tissue was collected at CF-LVAD implant and subsequently at transplant from 22 patients. Aortic wall morphometry and fibrillar collagen content (a measure of fibrosis) was quantified. In addition, whole-transcriptome profiling by RNA sequencing and follow-up immunohistochemistry were performed to evaluate CF-LVAD-mediated changes in aortic mRNA and protein expression. RESULTS: The mean age was 52 ± 12 years, with a mean duration of CF-LVAD of 224 ± 193 days (range 45-798 days). There was a significant increase in the thickness of the collagen-rich adventitial layer from 218 ± 110 µm pre-LVAD to 410 ± 209 µm post-LVAD (P < 0.01). Furthermore, there was an increase in intimal and medial mean fibrillar collagen intensity from 22 ± 11 a.u. pre-LVAD to 41 ± 24 a.u. post-LVAD (P < 0.0001). The magnitude of this increase in fibrosis was greater among patients with longer durations of CF-LVAD support. CF-LVAD led to profound down-regulation in expression of extracellular matrix-degrading enzymes, such as matrix metalloproteinase-19 and ADAMTS4, whereas no evidence of fibroblast activation was noted. CONCLUSIONS: There is aortic remodeling and fibrosis after CF-LVAD that correlates with the duration of support. This fibrosis is due, at least in part, to suppression of extracellular matrix-degrading enzyme expression. Further research is needed to examine the contribution of nonphysiological flow patterns on vascular function and whether modulation of pulsatility may improve vascular remodeling and long-term outcomes.

Simvastatin inhibits angiotensin II-induced abdominal aortic aneurysm formation in apolipoprotein E-knockout mice: possible role of ERK.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a life-threatening disease affecting almost 10% of the population over age 65. Generation of AAAs by infusion of angiotensin (Ang) II in apolipoprotein E-knockout (ApoE(-/-)) mice is an animal model which supports an imbalance of the renin-angiotensin system in the pathogenesis of AAA. The effect of statins on AngII-mediated AAA formation and the associated neovascularization is not known. Here we determined the effect of simvastatin and the ERK inhibitor, CI1040, on AngII-stimulated AAA formation. METHODS AND RESULTS: ApoE(-/-) mice infused for 28 days with AngII using osmotic minipumps were treated with placebo, 10 mg/kg/d simvastatin, or 100 mg/kg/d CI1040. 95% of AngII-treated mice developed AAA with neovascularization of the lesion, increased ERK phosphorylation, MCP-1 secretion, and MMP activity. These effects were markedly reversed by simvastatin and in part by CI1040. Furthermore, simvastatin and the ERK inhibitor U0126 reversed AngII-stimulated angiogenesis and MMP secretion by human umbilical vein endothelial cells. CONCLUSIONS: These data support the conclusion that simvastatin interferes with AAA formation induced by AngII in ApoE(-/-) mice at least in part via ERK inhibition.

Smooth muscle-derived progenitor cell myofibroblast differentiation through KLF4 downregulation promotes arterial remodeling and fibrosis.

Resident vascular adventitial SCA1+ progenitor (AdvSca1) cells are essential in vascular development and injury. However, the heterogeneity of AdvSca1 cells presents a unique challenge in understanding signaling pathways orchestrating their behavior in homeostasis and injury responses. Using smooth muscle cell (SMC) lineage-tracing models, we identified a subpopulation of AdvSca1 cells (AdvSca1-SM) originating from mature SMCs that undergo reprogramming in situ and exhibit a multipotent phenotype. Here we employed lineage tracing and RNA-sequencing to define the signaling pathways regulating SMC-to-AdvSca1-SM cell reprogramming and AdvSca1-SM progenitor cell phenotype. Unbiased hierarchical clustering revealed that genes related to hedgehog/WNT/beta-catenin signaling were significantly enriched in AdvSca1-SM cells, emphasizing the importance of this signaling axis in the reprogramming event. Leveraging AdvSca1-SM-specific expression of GLI-Kruppel family member GLI1 (Gli1), we generated Gli1-CreERT2-ROSA26-YFP reporter mice to selectively track AdvSca1-SM cells. We demonstrated that physiologically relevant vascular injury or AdvSca1-SM cell-specific Kruppel-like factor 4 (Klf4) depletion facilitated the proliferation and differentiation of AdvSca1-SM cells to a profibrotic myofibroblast phenotype rather than macrophages. Surprisingly, AdvSca1-SM cells selectively contributed to adventitial remodeling and fibrosis but little to neointima formation. Together, these findings strongly support therapeutics aimed at preserving the AdvSca1-SM cell phenotype as a viable antifibrotic approach.

Attenuated expression of profilin-1 confers protection from atherosclerosis in the LDL receptor null mouse.

Atherosclerosis-related events are a major cause of morbidity and death worldwide, but the mechanisms underlying atherogenesis are not fully understood. We showed in previous studies that the actin-binding protein profilin-1 (pfn) was upregulated in atherosclerotic plaques and in endothelial cells (ECs) treated with oxidized low-density lipoproteins (oxLDL). The present study addressed the role of pfn in atheroma formation. To this end, mice with heterozygous deficiency of pfn, Pfn(+/-), were crossed with Ldlr(-/-) mice. After 2 months under a 1.25% cholesterol atherogenic diet, Pfn(+/-)Ldlr(-/-) (PfnHet) exhibited a significant reduction in lesion burden compared with Ldlr(-/-) control mice (PfnWT), whereas total cholesterol and triglyceride levels were similar in the 2 groups. Relevant atheroprotective changes were identified in PfnHet. When compared with PfnWT, aortas from PfnHet mice showed preserved endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO)-dependent signaling, and reduced vascular cell adhesion molecule (VCAM)-1 expression and macrophage accumulation at lesion-prone sites. Similarly, knockdown of pfn in cultured aortic ECs was protective against endothelial dysfunction triggered by oxLDL. Finally, bone marrow-derived macrophages from PfnHet showed blunted internalization of oxLDL and oxLDL-induced inflammation. These studies demonstrate that pfn levels modulate processes critical for early atheroma formation and suggest that pfn heterozygosity confers atheroprotection through combined endothelial- and macrophage-dependent mechanisms.

Coronary Artery Remodeling and Fibrosis With Continuous-Flow Left Ventricular Assist Device Support.

BACKGROUND: Coronary artery fluid dynamics may be altered because of the nonphysiological flow seen in continuous-flow left ventricular assist devices (CF-LVADs). Our aim was to study the structure and composition of coronary vessels after CF-LVAD. METHODS AND RESULTS: Coronary arteries were collected from patients with heart failure (HF) at the time of transplantation, of whom 15 were supported with a CF-LVAD before transplant (HF+LVAD group) and 9 were not (HF non-LVAD group). In addition, coronary samples were obtained from 5 nonfailing age-matched donors (nonfailing group). Histological analysis was performed to quantify coronary morphology, composition, vascular fibrosis, and vasa vasorum density. The age and sex mix of the 3 groups were similar, and the mean duration of LVAD support was 213 days. Compared with patients with HF and nonfailing donors, the arteries from patients with HF+LVAD had expansion of the adventitia, breakdown of the internal elastic lamina, and increased adventitial collagen deposition and density of vasa vasorum. CONCLUSIONS: Among patients supported with CF-LVADs, the coronary arteries develop marked remodeling with increased adventitial fibrosis. The physiological consequences of these structural changes are unknown, but it is possible that arterial contractility may be impaired, thus limiting coronary flow reserve and promoting myocardial ischemia. This may contribute to CF-LVAD complications, such as ventricular arrhythmias and right ventricular failure. As more patients receive CF-LVADs and new pump technology attempts to modulate flow profiles and pulsatility, further research is needed to understand the mechanisms and long-term sequela of these changes in coronary arteries and other vascular beds.

PTEN deficiency promotes pathological vascular remodeling of human coronary arteries.

Phosphatase and tensin homolog (PTEN) is an essential regulator of the differentiated vascular smooth muscle cell (SMC) phenotype. Our goal was to establish that PTEN loss promotes SMC dedifferentiation and pathological vascular remodeling in human atherosclerotic coronary arteries and nonatherosclerotic coronary arteries exposed to continuous-flow left ventricular assist devices (CF-LVADs). Arteries were categorized as nonatherosclerotic hyperplasia (NAH), atherosclerotic hyperplasia (AH), or complex plaque (CP). NAH coronary arteries from CF-LVAD patients were compared to NAH coronaries from non-LVAD patients. Intimal PTEN and SMC contractile protein expression was reduced compared with the media in arteries with NAH, AH, or CP. Compared with NAH, PTEN and SMC contractile protein expression was reduced in the media and intima of arteries with AH and CP. NAH arteries from CF-LVAD patients showed marked vascular remodeling and reduced PTEN and α-smooth muscle actin (αSMA) in medial SMCs compared with arteries from non-LVAD patients; this correlated with increased medial collagen deposition. Mechanistically, compared with ApoE-/- mice, SMC-specific PTEN-null/ApoE-/- double-knockout mice exhibited accelerated atherosclerosis progression and increased vascular fibrosis. By microarray and validated quantitative RT-PCR analysis, SMC PTEN deficiency promotes a global upregulation of proinflammatory and profibrotic genes. We propose that PTEN is an antiinflammatory, antifibrotic target that functions to maintain SMC differentiation. SMC loss of PTEN results in pathological vascular remodeling of human arteries.

Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation.

Vascular disease progression is associated with marked changes in vascular smooth muscle cell (SMC) phenotype and function. SMC contractile gene expression and, thus differentiation, is under direct transcriptional control by the transcription factor, serum response factor (SRF); however, the mechanisms dynamically regulating SMC phenotype are not fully defined. Here we report that the lipid and protein phosphatase, PTEN, has a novel role in the nucleus by functioning as an indispensible regulator with SRF to maintain the differentiated SM phenotype. PTEN interacts with the N-terminal domain of SRF and PTEN-SRF interaction promotes SRF binding to essential promoter elements in SM-specific genes. Factors inducing phenotypic switching promote loss of nuclear PTEN through nucleo-cytoplasmic translocation resulting in reduced myogenically active SRF, but enhanced SRF activity on target genes involved in proliferation. Overall decreased expression of PTEN was observed in intimal SMCs of human atherosclerotic lesions underlying the potential clinical importance of these findings.

Loss of collagen XVIII enhances neovascularization and vascular permeability in atherosclerosis.

BACKGROUND: Plaque neovascularization is thought to promote atherosclerosis; however, the mechanisms of its regulation are not understood. Collagen XVIII and its proteolytically released endostatin fragment are abundant proteoglycans in vascular basement membranes and the walls of major blood vessels. We hypothesized that collagen XVIII in the aortic wall inhibits the proliferation and intimal extension of vasa vasorum. METHODS AND RESULTS: To test our hypothesis, we bred collagen XVIII-knockout (Col18a1(-/-)) mice into the atherosclerosis-prone apolipoprotein E-deficient (ApoE(-/-)) strain. After 6 months on a cholesterol diet, aortas from ApoE(-/-);Col18a1(-/-) and ApoE(-/-);Col18a1(+/-) heterozygote mice showed increased atheroma coverage and enhanced lipid accumulation compared with wild-type littermates. We observed more extensive vasa vasorum and intimal neovascularization in knockout but not heterozygote aortas. Endothelial cells sprouting from Col18a1(-/-) aortas were increased compared with heterozygote and wild-type aortas. In contrast, vascular permeability of large and small blood vessels was enhanced with even heterozygous loss of collagen XVIII but was not suppressed by increasing serum endostatin to wild-type levels. CONCLUSIONS: Our results identify a previously unrecognized function for collagen XVIII that maintains vascular permeability. Loss of this basement membrane proteoglycan enhances angiogenesis and vascular permeability during atherosclerosis by distinct gene-dose-dependent mechanisms.

Deletion of the basement membrane heparan sulfate proteoglycan type XVIII collagen causes hypertriglyceridemia in mice and humans.

BACKGROUND: Lipoprotein lipase (Lpl) acts on triglyceride-rich lipoproteins in the peripheral circulation, liberating free fatty acids for energy metabolism or storage. This essential enzyme is synthesized in parenchymal cells of adipose tissue, heart, and skeletal muscle and migrates to the luminal side of the vascular endothelium where it acts upon circulating lipoproteins. Prior studies suggested that Lpl is immobilized by way of heparan sulfate proteoglycans on the endothelium, but genetically altering endothelial cell heparan sulfate had no effect on Lpl localization or lipolysis. The objective of this study was to determine if extracellular matrix proteoglycans affect Lpl distribution and triglyceride metabolism. METHODS AND FINDINGS: We examined mutant mice defective in collagen XVIII (Col18), a heparan sulfate proteoglycan present in vascular basement membranes. Loss of Col18 reduces plasma levels of Lpl enzyme and activity, which results in mild fasting hypertriglyceridemia and diet-induced hyperchylomicronemia. Humans with Knobloch Syndrome caused by a null mutation in the vascular form of Col18 also present lower than normal plasma Lpl mass and activity and exhibit fasting hypertriglyceridemia. CONCLUSIONS: This is the first report demonstrating that Lpl presentation on the lumenal side of the endothelium depends on a basement membrane proteoglycan and demonstrates a previously unrecognized phenotype in patients lacking Col18.