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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Heterogeneous multi-laminar tissue constructs as a platform to evaluate aortic valve matrix-dependent pathogenicity.

Designing and constructing controlled in vitro cell culture platforms is imperative toward pinpointing factors that contribute to the development of calcific aortic valve disease. A 3D, laminar, filter paper-based cell culture system that was previously established as a method of analyzing valvular interstitial cell migration and protein expression was adapted here for studying the impact of specific extracellular matrix proteins on cellular viability and calcification proclivity. Hydrogels incorporating hyaluronan and collagen I, two prevalent valvular extracellular matrix proteins with altered pathological production, were designed with similar mechanics to parse out effects of the individual proteins on cell behavior. Laminar constructs containing varying combinations of discrete layers of collagen and hyaluronan were assembled to mimic native and pathological valve compositions. Proteinaceous and genetic expression patterns pertaining to cell viability and calcific potential were quantified via fluorescent imaging. A significant dose-dependency was observed, with increased collagen content associated with decreased viability and increased calcific phenotype. These results suggest that extracellular composition is influential in calcific aortic valve disease progression and will be key toward development of future tissue-engineered or pharmaceutical calcific aortic valve treatments. STATEMENT OF SIGNIFICANCE: Calcific aortic valve disease (CAVD), a widespread heart valve disorder, is characterized by fibrotic leaflet thickening and calcific nodule formation. This pathological remodeling is an active process mediated by the valvular interstitial cells (VICs). Currently, the only treatment available is surgical replacement of the valve - a procedure associated with significant long-term risk and morbidity. Development of effective alternate therapies is hindered by our poor understanding of CAVD etiology. Previous work has implicated the composition and mechanics of the extracellular matrix in the progression of CAVD. These individual factors and their magnitude of influence have not been extensively explored - particularly in 3D systems. Here, we have bridged this gap in understanding through the employment of a heterogeneous 3D filter-paper culture system.

Differentiation of spontaneously contracting cardiomyocytes from non-virally reprogrammed human amniotic fluid stem cells.

Congenital heart defects are the most common birth defect. The limiting factor in tissue engineering repair strategies is an autologous source of functional cardiomyocytes. Amniotic fluid contains an ideal cell source for prenatal harvest and use in correction of congenital heart defects. This study aims to investigate the potential of amniotic fluid-derived stem cells (AFSC) to undergo non-viral reprogramming into induced pluripotent stem cells (iPSC) followed by growth-factor-free differentiation into functional cardiomyocytes. AFSC from human second trimester amniotic fluid were transfected by non-viral vesicle fusion with modified mRNA of OCT4, KLF4, SOX2, LIN28, cMYC and nuclear GFP over 18 days, then differentiated using inhibitors of GSK3 followed 48 hours later by inhibition of WNT. AFSC-derived iPSC had high expression of OCT4, NANOG, TRA-1-60, and TRA-1-81 after 18 days of mRNA transfection and formed teratomas containing mesodermal, ectodermal, and endodermal germ layers in immunodeficient mice. By Day 30 of cardiomyocyte differentiation, cells contracted spontaneously, expressed connexin 43 and ß-myosin heavy chain organized in sarcomeric banding patterns, expressed cardiac troponin T and ß-myosin heavy chain, showed upregulation of NKX2.5, ISL-1 and cardiac troponin T with downregulation of POU5F1, and displayed calcium and voltage transients similar to those in developing cardiomyocytes. These results demonstrate that cells from human amniotic fluid can be differentiated through a pluripotent state into functional cardiomyocytes.