Arterial Extracellular Matrix Research Articles

Fabrication and mechanical characterization of near field electrospun bioresorbable vascular grafts with fibrous architecture mimicking the arterial extracellular matrix

Cardiovascular disease, arteriosclerosis, is characterized by the thickening of blood vessel (arteries) walls restricting blood flow and is a global health problem. One treatment option is a surgical procedure utilizing an autologous or synthetic vascular graft to bypass or replace the diseased arterial segment. The goal of this study was to fabricate and mechanically characterize near field electrospun bioresorbable vascular grafts with a fibrous architecture that mimics the arterial extracellular matrix. Polydioxanone vascular constructs with circumferential fiber alignment angles of 15°/75° and 30°/60° (0° representing circumferential fiber alignment) were fabricated using a custom built near-field electrospinning (NFES) system. The vascular construct mechanical properties were compared to the saphenous vein (SV) and internal mammary artery (IMA) through longitudinal and circumferential uniaxial mechanical testing, suture retention, and burst pressure evaluations. The results demonstrated that the 15°/75° templates were closest to mimicking the native vessel target properties as compared to the 30°/60°; however, neither of the vascular template designs achieved or exceeded all the target values. For the ultimate tensile strength, both the constructs met the SV value on the circumferential axis (2.61 MPa) and the IMA value on the longitudinal axis (4.3 MPa). In terms of suture retention, the 15°/75° template was the only construct that was in the IMA and SV targeted range of 138–200 gf. Finally, the burst pressure testing results indicated that neither of the vascular constructs achieved the level of the SV or IMA (1600–3196 mmHg), however, the 15° constructs were within the lower error of the SV. In conclusion, with further design modifications, NFES constructs have demonstrated promise as small-diameter vascular grafts by mimicking native arterial architecture and mechanical properties.

Cell contractility and Focal Adhesion Kinase control circumferential arterial stiffness.

Arterial stiffening is a hallmark of aging and cardiovascular disease. While it is well established that vascular smooth muscle cells (SMCs) contribute to arterial stiffness by synthesizing and remodeling the arterial extracellular matrix, the direct contributions of SMC contractility and mechanosensors to arterial stiffness, and particularly the arterial response to pressure, remain less well understood despite being a long-standing question of biomedical importance. Here, we have examined this issue by combining use of pressure myography of intact carotid arteries, pharmacologic inhibition of contractility, and genetic deletion of SMC focal adhesion kinase (FAK). Biaxial inflation-extension tests performed at physiological pressures showed that acute inhibition of cell contractility with blebbistatin or EGTA altered vessel geometry and preferentially reduced circumferential, as opposed to axial, arterial stiffness in wild-type mice. Similarly, genetic deletion of SMC FAK, which attenuated arterial contraction to KCl, reduced vessel wall thickness and circumferential arterial stiffness in response to pressure while having minimal effect on axial mechanics. Moreover, these effects of FAK deletion were lost by treating arteries with blebbistatin or by inhibiting myosln light chain kinase. The expression of arterial fibrillar collagens, the integrity of arterial elastin, or markers of SMC differentiation were not affected by deletion of SMC FAK. Our results connect cell contractility and SMC FAK to the regulation of arterial wall thickness and directionally-specific arterial stiffening.

Pulmonary hypertension: Linking inflammation and pulmonary arterial stiffening.

Pulmonary hypertension (PH) is a progressive disease that arises from multiple etiologies and ultimately leads to right heart failure as the predominant cause of morbidity and mortality. In patients, distinct inflammatory responses are a prominent feature in different types of PH, and various immunomodulatory interventions have been shown to modulate disease development and progression in animal models. Specifically, PH-associated inflammation comprises infiltration of both innate and adaptive immune cells into the vascular wall of the pulmonary vasculature—specifically in pulmonary vascular lesions—as well as increased levels of cytokines and chemokines in circulating blood and in the perivascular tissue of pulmonary arteries (PAs). Previous studies suggest that altered hemodynamic forces cause lung endothelial dysfunction and, in turn, adherence of immune cells and release of inflammatory mediators, while the resulting perivascular inflammation, in turn, promotes vascular remodeling and the progression of PH. As such, a vicious cycle of endothelial activation, inflammation, and vascular remodeling may develop and drive the disease process. PA stiffening constitutes an emerging research area in PH, with relevance in PH diagnostics, prognostics, and as a therapeutic target. With respect to its prognostic value, PA stiffness rivals the well-established measurement of pulmonary vascular resistance as a predictor of disease outcome. Vascular remodeling of the arterial extracellular matrix (ECM) as well as vascular calcification, smooth muscle cell stiffening, vascular wall thickening, and tissue fibrosis contribute to PA stiffening. While associations between inflammation and vascular stiffening are well-established in systemic vascular diseases such as atherosclerosis or the vascular manifestations of systemic sclerosis, a similar connection between inflammatory processes and PA stiffening has so far not been addressed in the context of PH. In this review, we discuss potential links between inflammation and PA stiffening with a specific focus on vascular calcification and ECM remodeling in PH.

Physiological basis for longitudinal motion of the arterial wall.

As opposed to arterial distension in the radial plane, longitudinal wall motion (LWM) is a multiphasic and bidirectional displacement of the arterial wall in the anterograde (i.e., in the direction of blood flow) and retrograde (i.e., opposing direction of blood flow) directions. Although initially disregarded as imaging artifact, LWM has been consistently reported in ultrasound investigations in the past decade and is reproducible beat-to-beat, albeit with large interindividual variability across healthy and diseased populations. Emerging literature has sought to examine the mechanistic control of LWM to explain the shape and variability of the motion pattern but lacks considerations for key foundational vascular principles at the level of the arterial wall ultrastructure. The purpose of this review is to summarize the potential factors that underpin the causes and control of arterial LWM, spanning considerations from the arterial extracellular matrix to systems-level integrative theories. First, an overview of LWM and relevant aspects wall composition will be discussed, including major features of the multiphasic pattern, arterial wall extracellular components, tunica fiber orientations, and arterial longitudinal prestretch. Second, current theories on the systems-level physiological mechanisms driving LWM will be discussed in the context of available evidence including experimental human research, porcine studies, and mathematical models. Throughout, we discuss implications of these observations with suggestions for future priority research areas.

Hypoxia of human endothelial artery wall cells affects arterial extracellular matrix remodelling and contributes to atherosclerosis development

Hypoxia of human endothelial artery wall cells affects arterial extracellular matrix remodelling and contributes to atherosclerosis development

X-ray microtomography reveals a lattice-like network within aortic elastic lamellae.

The arterial wall consists of three concentric layers: intima, media, and adventitia. Beyond their resident cells, these layers are characterized by an extracellular matrix (ECM), which provides both biochemical and mechanical support. Elastin, the major component of arterial ECM, is present in the medial layer and organized in concentric elastic lamellae that confer resilience to the wall. We explored the arterial wall structures from C57Bl6 (control), db/db (diabetic), and ApoE-/- (atherogenic) mice aged 3months using synchrotron X-ray computed microtomography on fixed and unstained tissues with a large image field (8 mm3 ). This approach combined a good resolution (0.83µm/voxel), large 3D imaging field. and an excellent signal to noise ratio conferred by phase-contrast imaging. We determined from 2D virtual slices that the thickness of intramural ECM structures was comparable between strains but automated image analysis of the 3D arterial volumes revealed a lattice-like network within concentric elastic lamellae. We hypothesize that this network could play a role in arterial mechanics. This work demonstrates that phase-contrast synchrotron X-ray computed microtomography is a powerful technique which to characterize unstained soft tissues.

Soluble epoxide hydrolase deletion attenuated nicotine-induced arterial stiffness via limiting the loss of SIRT1.

Arterial stiffness, a consequence of smoking, is an underlying risk factor of cardiovascular diseases. Epoxyeicosatrienoic acids (EETs), hydrolyzed by soluble epoxide hydrolase (sEH), have beneficial effects against vascular dysfunction. However, the role of sEH knockout in nicotine-induced arterial stiffness was not characterized. We hypothesized that sEH knockout could prevent nicotine-induced arterial stiffness. In the present study, Ephx2 (the gene encodes sEH enzyme) null (Ephx2-/-) mice and wild-type (WT) littermate mice were infused with or without nicotine and administered with or without nicotinamide [NAM, sirtuin-1 (SIRT1) inhibitor] simultaneously for 4 wk. Nicotine treatment increased sEH expression and activity in the aortas of WT mice. Nicotine infusion significantly induced vascular remodeling, arterial stiffness, and SIRT1 deactivation in WT mice, which was attenuated in Ephx2 knockout mice (Ephx2-/- mice) without NAM treatment. However, the arterial protective effects were gone in Ephx2-/- mice with NAM treatment. In vitro, 11,12-EET treatment attenuated nicotine-induced matrix metalloproteinase 2 (MMP2) upregulation via SIRT1-mediated yes-associated protein (YAP) deacetylation. In conclusion, sEH knockout attenuated nicotine-induced arterial stiffness and vascular remodeling via SIRT1-induced YAP deacetylation.NEW & NOTEWORTHY We presently show that sEH knockout repressed nicotine-induced arterial stiffness and extracellular matrix remodeling via SIRT1-induced YAP deacetylation, which highlights that sEH is a potential therapeutic target in smoking-induced arterial stiffness and vascular remodeling.

Elastic tissue disruption is a major pathogenic factor to human vascular disease.

Elastic fibers are essential components of the arterial extracellular matrix. They consist of the protein elastin and an array of microfibrils that support the protein and connect it to the surrounding matrix. The elastin gene encodes tropoelastin, a protein that requires extensive cross-linking to become elastin. Tropoelastin is expressed throughout human life, but its expression levels decrease with age, suggesting that the potential to synthesize elastin persists during lifetime although declines with aging. The initial abnormality documented in human atherosclerosis is fragmentation and loss of the elastic network in the medial layer of the arterial wall, suggesting an imbalance between elastic fiber injury and restoration. Damaged elastic structures are not adequately repaired by synthesis of new elastic elements. Progressive collagen accumulation follows medial elastic fiber disruption and fibrous plaques are formed, but advanced atherosclerosis lesions do not develop in the absence of prior elastic injury. Aging is associated with arterial extracellular matrix anomalies that evoke those present in early atherosclerosis. The reduction of elastic fibers with subsequent collagen accumulation leads to arterial stiffening and intima-media thickening, which are independent predictors of incident hypertension in prospective community-based studies. Arterial stiffening precedes the development of hypertension. The fundamental role of the vascular elastic network to arterial structure and function is emphasized by congenital disorders caused by mutations that disrupt normal elastic fiber production. Molecular changes in the genes coding tropoelastin, lysyl oxidase (tropoelastin cross-linking), and elastin-associated microfibrils, including fibrillin-1, fibulin-4, and fibulin-5 produce severe vascular injury due to absence of functional elastin.

Mechanosensitive smooth muscle cell phenotypic plasticity emerging from a null state and the balance between Rac and Rho.

SUMMARYReversible differentiation of vascular smooth muscle cells (VSMCs) plays a critical role in vascular biology and disease. Changes in VSMC differentiation correlate with stiffness of the arterial extracellular matrix (ECM), but causal relationships remain unclear. We show that VSMC plasticity is mechanosensitive and that both the de-differentiated and differentiated fates are promoted by the same ECM stiffness. Differential equations developed to model this behavior predicted that a null VSMC state generates the dual fates in response to ECM stiffness. Direct measurements of cellular forces, proliferation, and contractile gene expression validated these predictions and showed that fate outcome is mediated by Rac-Rho homeostasis. Rac, through distinct effects on YAP and TAZ, is required for both fates. Rho drives the contractile state alone, so its level of activity, relative to Rac, drives phenotypic choice. Our results show how the cellular response to a single ECM stiffness generates bi-stability and VSMC plasticity.

Elevation of Serum Cystatin C Levels as a Predictor of Cardiovascular Risk in Type 2 Diabetes Mellitus Patients: A Case-control Study

Introduction: Patients with type 2 Diabetes Mellitus (DM) exhibit atherogenic lipid profile, increasing the risk for atherosclerosis, compared to people without diabetes. Cystatin C, a naturally occurring protease inhibitor has a key role in pathogenesis of atherosclerosis in type 2 DM patients by inhibiting the elastolytic activity of arterial extracellular matrix by cysteine protease cathepsins. Increased concentration of cystatin C in serum reflects a counterbalance of the damaging elastolytic activity which is involved in the pathogenesis of atherosclerosis in type 2 DM patients. Aim: To determine the serum levels of cystatin C and lipid profile in type 2 DM patients with good and poor glycaemic control and to find out the correlation between serum cystatin C with HbA1c levels and cardiovascular risk markers (non-High-Density Lipoprotein Cholesterol (HDLc), Total Cholesterol (TC)/HDLc, Low-Density Liporotein (LDLc)/HDLc). Materials and Methods: The present case-control study included 60 diabetic patients of 35-75 years of age including both the genders. They were divided into Group A (n=30) with HbA1c ≤6.5% and Group B (n=30) with HbA1c >6.5%. Blood samples were collected in fasting state and analysed for Fasting Blood Sugar (FBS), TC, HDLc, LDLc. FBS was analysed by Hexokinase method, HbA1c by turbidimetric inhibition immunoassay and cystatin C by Immunoturbidimetric method. Other parameters like Non-HDLc and cardiovascular risk ratios TC/HDLc and LDLc/HDLc were calculated. Statistical analysis was done by student’s t-test and statistical significance was established at p-value <0.0001. Pearson’s correlation coefficient was used to examine various parameters. Results: In the present study, it was found that diabetic patients with HbA1c >6.5% showed increased levels of serum cystatin C (1.20±0.73 mg/L), TC (182±46.48 mg/dL), LDLc (137±13.86 mg/dL) and low levels of HDLc (34.7±12.43 mg/dL) compared to diabetic patients with HbA1c ≤6.5%. Increase in serum cystatin C levels correlated well with the increased levels of HbA1c (r=0.92), non-HDLc (r=0.87) and cardiovascular risk ratios like TC/HDLc (r=0.95) and LDLc/HDLc (r=0.97). Conclusion: The elevated serum levels of cystatin C levels correlated well with increased HbA1c and lipid profile indicating the dyslipidemia and proatherogenic status in type 2 diabetic patients with poor glycaemic control. Thus, cystatin C can be considered as the predictive marker for cardiovascular disease in type 2 diabetic patients.

Collagen remodeling markers show differentiated expression in patients with ST- and non-ST elevation myocardial infarction

Abstract Background Following acute myocardial infarction (MI), the left ventricle undergoes molecular and extracellular matrix (ECM) changes. The ECM is a dynamic structure with a potential role in cardiac remodeling post-MI. Collagens are the major components of both cardiac and arterial ECM. Purpose We evaluated circulating levels of type I, IV and VI collagen fragments in two cohorts of patients with acute MI to investigate collagen turnover post-MI. The cohorts were Malmö AMI in elderly (MAMI-Y) and Assessing Platelet Activity in Coronary Heart Disease (APACHE). Methods Serum was collected from 190 patients from the discovery cohort (MAMI-Y: mean age 74, SD 10.8) at four timepoints: admission when MI, after 3–6 days, 6 weeks, 12 months; citrate plasma was collected from 142 patients from the validation cohort (APACHE: mean age 65, SD 11.6) at four timepoints: hospitalization, 3 days, 7–9 days, 6 months. The biomarkers of matrix metalloproteinase (MMP)-mediated degradation of type I collagen (C1M), MMP-mediated degradation of type IV collagen (C4M) and formation of type VI collagen (PRO-C6) were measured at all timepoints (immunosorbent assays). Differences in the markers at the different timepoints were calculated using repeated measures ANOVA. Results Circulating levels of the formation biomarker PRO-C6 significantly increased from baseline and remained high at all three following timepoints in both MAMI-Y and APACHE studies (all p<0.001). In contrast, the degradation biomarkers C1M and C4M showed a similar pattern of an initial increase 3 days post-MI followed by a decrease over time, with C1M in MAMI-Y and both C1M and C4M in APACHE having returned to baseline level by the final timepoint. Circulating baseline levels of PRO-C6 correlated with age (r=0.397, p<0.0001 in MAMI-Y, r=0.427, p<0.0001 in APACHE). Categorizing the subjects into ST elevation MI (STEMI; MAMI-Y: N=67, APACHE: N=71) or non-ST elevation MI (NSTEMI; MAMI-Y: N=130, APACHE: N=52) revealed that a correlation with age remained in both subgroups (r=0.443, p<0.0001 and r=0.325, p<0.0001, respectively, in MAMI-Y, and r=0.516, p<0.0001 and r=0.316, p=0.023, respectively, in APACHE). Moreover, PRO-C6 was elevated in STEMI patients that had previously experienced an MI in both cohorts (MAMI-Y: p=0.017, APACHE: p=0.016). C1M and C4M levels were not different in patients with prior MI in any of the cohorts. No association was found between any biomarker and gender or diabetes. Echocardiography showed a correlation between baseline levels of C1M and ejection fraction (r=0.228, p=0.023) in the whole MAMI-Y cohort and among NSTEMI, but not among STEMI, subjects (r=0.337, p=0.004). Conclusions We observed changes in circulating fragments reflecting collagen turnover in the acute phase post-MI, more pronounced in STEMI patients. This may indicate that STEMI patients have more active collagen remodeling than NSTEMI patients and may have more altered left ventricle function and remodeling. Funding Acknowledgement Type of funding source: Other. Main funding source(s): This work was supported by the Danish Research Foundation “den danske forskningsfond”, the Innovation foundation (Innovationsfonden), Swedish Research Council, Swedish Heart and Lung Foundation, Swedish Society for Medical Research, Swedish Society of Medicine, the Crafoord Foundation, the Åke Wiberg foundation and the Stroke foundation.

Extracellular vesicles influence the pulmonary arterial extracellular matrix in congenital diaphragmatic hernia.

Abnormal pulmonary vasculature directly affects the development and progression of congenital diaphragmatic hernia (CDH)-associated pulmonary hypertension (PH). Though overarching structural and cellular changes in CDH-affected pulmonary arteries have been documented, the precise role of the extracellular matrix (ECM) in the pulmonary artery (PA) pathophysiology remains undefined. Here, we quantify the structural, compositional, and mechanical CDH-induced changes in the main and distal PA ECM and investigate the efficacy of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) as a therapy to ameliorate pathological vascular ECM changes. Pregnant Sprague-Dawley rodents were administered nitrofen to induce CDH-affected pulmonary vasculature in the offspring. A portion of CDH-affected pups was treated with intravenous infusion of MSC-EVs (1 × 1010 /mL) upon birth. A suite of histological, mechanical, and transmission electron microscopic analyses were utilized to characterize the PA ECM. The CDH model main PA presented significantly altered characteristics-including greater vessel thickness, greater lysyl oxidase (LOX) expression, and a relatively lower ultimate tensile strength of 13.6 MPa compared to control tissue (25.1 MPa), suggesting that CDH incurs ECM structural disorganization. MSC-EV treatment demonstrated the potential to reverse CDH-related changes, particularly through rapid inhibition of ECM remodeling enzymes (LOX and MMP-9). Additionally, MSC-EV treatment bolstered structural aspects of the PA ECM and mitigated pathological disorganization as exhibited by increased medial wall thickness and stiffness that, while not significantly altered, trends away from CDH-affected tissue. These data demonstrate notable ECM remodeling in the CDH pulmonary vasculature, along with the capacity of MSC-EVs to attenuate pathological ECM remodeling, identifying MSC-EVs as a potentially efficacious therapeutic for CDH-associated pulmonary hypertension.

Extracellular Matrix Analysis of Human Renal Arteries in Both Quiescent and Active Vascular State.

In vascular tissue engineering strategies, the addition of vascular-specific extracellular matrix (ECM) components may better mimic the in vivo microenvironment and potentially enhance cell–matrix interactions and subsequent tissue growth. For this purpose, the exact composition of the human vascular ECM first needs to be fully characterized. Most research has focused on characterizing ECM components in mature vascular tissue; however, the developing fetal ECM matches the active environment required in vascular tissue engineering more closely. Consequently, we characterized the ECM protein composition of active (fetal) and quiescent (mature) renal arteries using a proteome analysis of decellularized tissue. The obtained human fetal renal artery ECM proteome dataset contains higher levels of 15 ECM proteins versus the mature renal artery ECM proteome, whereas 16 ECM proteins showed higher levels in the mature tissue compared to fetal. Elastic ECM proteins EMILIN1 and FBN1 are significantly enriched in fetal renal arteries and are mainly produced by cells of mesenchymal origin. We functionally tested the role of EMILIN1 and FBN1 by anchoring the ECM secreted by vascular smooth muscle cells (SMCs) to glass coverslips. This ECM layer was depleted from either EMILIN1 or FBN1 by using siRNA targeting of the SMCs. Cultured endothelial cells (ECs) on this modified ECM layer showed alterations on the transcriptome level of multiple pathways, especially the Rho GTPase controlled pathways. However, no significant alterations in adhesion, migration or proliferation were observed when ECs were cultured on EMILIN1- or FNB1-deficient ECM. To conclude, the proteome analysis identified unique ECM proteins involved in the embryonic development of renal arteries. Alterations in transcriptome levels of ECs cultured on EMILIN1- or FBN1-deficient ECM showed that these candidate proteins could affect the endothelial (regenerative) response.

Porcine arterial ECM hydrogel: Designing an in vitro angiogenesis model for long-term high-throughput research.

The field of angiogenesis research provides deep understanding regarding this important process, which plays fundamental roles in tissue development and different abnormalities. In vitro models offer the advantages of low-cost high-throughput research of angiogenesis while sparing animal lives, and enabling the use of human cells. Nevertheless, prevailing in vitro models lack stability and are limited to a few days' assays. This study, therefore, examines the hypothesis that closely mimicking the vascular microenvironment can more reliably support longer angiogenesis processes in vitro. To this end, porcine arterial extracellular matrix (paECM)- a key component of blood vessels-was isolated and processed into a thermally induced hydrogel and characterized in terms of composition, structure, and mechanical properties, thus confirming the preservation of important characteristics of arterial extracellular matrix. This unique hydrogel was further tailored into a three-dimensional model of angiogenesis using endothelial cells and supporting cells, in a configuration that allows high-throughput quantitative analysis of cell viability and proliferation, cell migration, and apoptosis, thus revealing the advantages of paECM over frequently used biomaterials. Markedly, when applied with well-known effectors of angiogenesis, the model measures reflected the expected response, hence validating its efficacy and establishing its potential as a promising tool for the research of angiogenesis.

Serum MMP‐3 and its Association with Large Artery Stiffness

Large artery stiffness is an independent predictor of cardiovascular disease (CVD) and all‐cause mortality. Stiffening of the large arteries (e.g., the aorta) is characterized by a marked reduction in the elastin‐collagen ratio in the extracellular matrix (ECM) of the arterial wall, and is largely the result of fatigue and fragmentation of ECM components due to cyclic stress. Matrix metalloproteinases (MMPs), a family of zinc‐dependent endopeptidases, may be important in the progression of arterial stiffness due to their involvement in ECM homeostasis and arterial wall remodeling. MMP‐3 may be of particular importance in arterial wall remodeling due to its ability to degrade numerous constituents of the arterial ECM, such as elastin and collagen. Previous studies have examined the effects of MMP‐3 genotype and expression on arterial wall stiffness in different disease populations; however, none have examined the association between MMP‐3 expression and large artery stiffness in a population of healthy young adults. Thus, the purpose of this study was to examine the association between serum MMP‐3 and carotid‐femoral pulse wave velocity (cfPWV), a non‐invasive measure of large artery stiffness, in a sample of healthy young adults. It is expected that individuals with higher serum MMP‐3 levels will present with larger cfPWVs. 156 participants (n = 68 males) aged 20–25 years were recruited as part of the Niagara Longitudinal Heart Study (NLHS), and all participants were free of any clinically diagnosed CVD. cfPWV (m/s) was determined using applanation tonometry as a non‐invasive surrogate of large artery stiffness. Serum MMP‐3 concentrations (pg/mL) were measured using standard ELISA techniques. Linear regression analyses were used to investigate the cross‐sectional association between serum MMP‐3 and cfPWV. Analyses were adjusted for age, sex, mean arterial pressure (MAP), body mass index (BMI), and smoking status. Data on cfPWV and MMP‐3 were available on 139 participants (n = 63 males), and were subsequently used in analysis. After adjustment for age, sex, MAP, BMI, and smoking status, serum MMP‐3 was significantly and positively associated with cfPWV (p = 0.038). cfPWV was also significantly and positively associated with MAP and smoking status (both p < 0.001), but not age (p = 0.336). The association between cfPWV and both sex and BMI showed a positive trend, but only reached borderline significance (p = 0.059 and p = 0.054, respectively). Together, MAP, smoking status and serum MMP‐3 predicted 22% of the variation in cfPWV (adjusted R2 = 0.220, p < 0.001). These data suggest that greater serum MMP‐3 levels are associated with larger cfPWVs and thus, greater large artery stiffness. Physiologic MMP‐3 levels function to maintain ECM homeostasis; however, greater MMP‐3 levels may exacerbate ECM degradation and contribute to stiffening of the large arteries. Future research should examine the potential functional role of MMP‐3 in CVD progression.Support or Funding InformationThe NLHS is funded by the Canadian Institute of Health Research (CIHR #363774 and #399332). KSD is funded by CIHR Doctoral Research Award—Frederick Banting and Charles Best Canada Graduate scholarship (RFN #167014). ARRM is supported by the Ontario Graduate Scholarship program

Experimental Investigation of Profibrotic Cell Signaling in Pulmonary Arterial Adventitial Fibroblasts

Pulmonary arterial adventitial fibroblasts (PAAFs) are important regulators of fibrotic vascular remodeling during pulmonary arterial hypertension (PAH). PAAFs can be activated into myofibroblasts that produce more extracellular matrix (ECM), which contributes significantly to pulmonary vascular remodeling and stiffening in disease progression. There are currently no therapies that reverse the matrix remodeling and vascular stiffening that occurs during PAH. In order to better identify treatments to target adventitial fibrosis, we sought to investigate the interplay between major pathways that have been reported to regulate myofibroblast transformation and ECM expression in PAAFs.Compared with other fibroblast types, there is a scarcity of experimental data on mechano‐signaling in PAAFs, therefore more in vitro experiments are needed to determine which pathways are crucial to creating a systems biology model that describes mechanosignaling in PAAFs.We have developed techniques to decellularize and reseed pulmonary arteries from Sprague‐Dawley rats to investigate the effects that purely the ECM have on the phenotypic transition of PAAFs. We surmise that pulmonary artery ECM alone from rats with PAH will have a significant impact on the transition of PAAFs into myofibroblasts and will investigate this difference using quantitative RT‐PCR. We will measure collagen I, collagen III expression, and alpha smooth muscle actin to measure the level of myofibroblast transition. In addition, levels of matrix proteins such as elastin and fibronectin will be measured via histology to characterize the level of fibrosis.Combining these results with a profibrotic signaling network model, we can use these methods to identify potential combinatorial therapeutics to prevent or inhibit wall remodeling to treat PAH.Support or Funding InformationThis work was partially funded by the American Heart Association Scientist Development Grant 16SDG29670010, and the National Institutes of Health NHLBI 1 T32 HL105373 and NHLBI 1 R01 HL137100.

Survivin is a Mechanosensitive Regulator of Vascular Smooth Muscle Cell Proliferation

Arterial stiffness induces pathological vascular smooth muscle cell (VSMC) phenotypes, causing atherogenesis and arterial stenosis when rigid arterial extracellular matrix signals VSMCs to aberrantly proliferate. Survivin is an inhibitor of apoptosis but alternatively promotes cell proliferation, and mounting evidence points to survivin inhabiting a key role in vascular injury and stiffening responses. Thus, we seek to understand if survivin plays a role in how mechanical forces drive the pathological proliferation of VSMCs.

Chemical stabilization of the extracellular matrix attenuates growth of experimentally induced abdominal aorta aneurysms in a large animal model

ObjectiveThe goal of the present study was to test the safety and efficacy of chemical stabilization of the arterial extracellular matrix as a novel nonoperative treatment of abdominal aortic aneurysms (AAAs) in a clinically relevant large animal model. MethodsTo achieve matrix stabilization, we used 1,2,3,4,6-pentagalloylglucose (PGG), a noncytotoxic polyphenolic agent capable of binding to and stabilizing elastin and collagen against the action of degrading enzymes. We first optimized the therapeutic PGG formulation and time of exposure by in vitro testing on porcine aortas using phenol histologic staining with iron chloride, elastic recoil assays, and PGG quantification as a function of tissue thickness. We then induced AAAs in 16 swine using sequential balloon angioplasty and elastase/collagenase and calcium chloride treatment of the infrarenal segment. We monitored AAA induction and development using digital subtraction angiography. At 2 weeks after induction, after the AAAs had reached ∼66% arterial expansion, the swine were randomly assigned to 2 groups. In the treatment group, we delivered PGG to the aneurysmal aorta endoluminally using a weeping balloon and evaluated the AAA diameters using digital subtraction angiography for another 10 weeks. The control swine did not receive any treatment. For the safety evaluation, we collected blood and performed comprehensive metabolic panels and complete blood counts every 2 to 3 weeks for all the animals. The swine were routinely monitored for neurologic and physical attributes such as behavior, inactivity, alertness, appetite, discomfort, and weight gain. After euthanasia and full necropsy, we analyzed the AAA tissue samples for PGG content, elastic recoil, and histologic features. ResultsIn vitro, a single 2.5-minute intraluminal delivery of 0.3% PGG to the swine aorta was sufficient for PGG to diffuse through the entire thickness of the porcine arterial tissues and to bind with high affinity to the elastic lamellae, as seen by positive iron chloride staining, a reduction of elastic recoil, and an increase in PGG content. In vivo, the control swine AAA tissues were thickened and showed the typical aspects of AAA, including chronic inflammation, adventitial reactivity, smooth muscle cell proliferation, elastic lamellae degradation, and medial and adventitial calcification. Similar aspects were noted in the PGG-treated arteries, except for the lack of calcification and an apparent diminished hyperplasia. PGG treatment was effective in reducing AAA expansion and reversing the process of AAA dilation by reducing the aortic diameters to ≤30% by week 12 (P < .05). PGG was specifically localized to the aneurysmal segments as seen by histologic examination, the reduction of elastic recoil, and an increase in PGG content. PGG treatment did not affect the swine's neurologic or physical attributes, weight, blood chemistry, blood cells, or functionality of remote organs. The control, untreated swine exhibited progressive increases in AAA diameters up to a mean value of 104%. ConclusionsLocalized delivery of PGG to the aneurysmal aorta attenuated AAA growth and reversed the course of the disease in the swine AAA model. Such specificity for diseased tissue is unprecedented in nonoperative AAA treatment. This novel paradigm-shifting approach has the potential to revolutionize AAA management and save thousands of lives.

Biomechanical Restoration Potential of Pentagalloyl Glucose after Arterial Extracellular Matrix Degeneration.

The objective of this study was to quantify pentagalloyl glucose (PGG) mediated biomechanical restoration of degenerated extracellular matrix (ECM). Planar biaxial tensile testing was performed for native (N), enzyme-treated (collagenase and elastase) (E), and PGG (P) treated porcine abdominal aorta specimens (n = 6 per group). An Ogden material model was fitted to the stress–strain data and finite element computational analyses of simulated native aorta and aneurysmal abdominal aorta were performed. The maximum tensile stress of the N group was higher than that in both E and P groups for both circumferential (43.78 ± 14.18 kPa vs. 10.03 ± 2.68 kPa vs. 13.85 ± 3.02 kPa; p = 0.0226) and longitudinal directions (33.89 ± 8.98 kPa vs. 9.04 ± 2.68 kPa vs. 14.69 ± 5.88 kPa; p = 0.0441). Tensile moduli in the circumferential direction was found to be in descending order as N > P > E (195.6 ± 58.72 kPa > 81.8 ± 22.76 kPa > 46.51 ± 15.04 kPa; p = 0.0314), whereas no significant differences were found in the longitudinal direction (p = 0.1607). PGG binds to the hydrophobic core of arterial tissues and the crosslinking of ECM fibers is one of the possible explanations for the recovery of biomechanical properties observed in this study. PGG is a beneficial polyphenol that can be potentially translated to clinical practice for preventing rupture of the aneurysmal arterial wall.

Activation of immune responses against the basement membrane component collagen type IV does not affect the development of atherosclerosis in ApoE-deficient mice

Oxidation of low-density lipoprotein (LDL) in the arterial extracellular matrix results in malondialdehyde (MDA)-modifications of surrounding matrix proteins. We have recently demonstrated an association between high levels of autoantibodies against MDA-modified collagen type IV and risk for development of myocardial infarction. Collagen type IV is an important component of the endothelial basement membrane and influences smooth muscle cell function. We hypothesized that immune responses against collagen type IV could contribute to vascular injury affecting the development of atherosclerosis. To investigate this possibility, we induced an antibody-response against collagen type IV in apolipoprotein E (Apo E)-deficient mice. Female ApoE−/− mice on C57BL/6 background were immunized with α1α2 type IV collagen chain peptides linked to the immune-enhancer PADRE, PADRE alone or PBS at 12 weeks of age with three subsequent booster injections before the mice were killed at 23 weeks of age. Immunization of PADRE alone induced autoantibodies against PADRE, increased IL-4 secretion from splenocytes and reduced SMC content in the subvalvular plaques. Immunization with peptides of α1α2 type IV collagen chains induced a strong IgG1antibody response against collagen type IV peptides without affecting the distribution of T cell populations, plasma cytokine or lipid levels. There were no differences in atherosclerotic plaque development between collagen α1α2(IV)-PADRE immunized mice and control mice. Our findings demonstrate that the presence of antibodies against the basement membrane component collagen type IV does not affect atherosclerosis development in ApoE−/− mice. This suggests that the association between autoantibodies against collagen type IV and risk for myocardial infarction found in humans does not reflect a pathogenic role of these autoantibodies.