Zannatul Ferdous

Strain Magnitude-Dependent Calcific Marker Expression in Valvular and Vascular Cells

Aortic valve disease and atherosclerosis tend to coexist in most patients with cardiovascular disease; however, the causes and mechanisms of disease development in heart valves are still not clearly understood. To understand the contributions of the magnitude of cyclic strain (5% hypotension, 10% physiological, and 15% hypertension) in calcification, we used a model system of tissue-engineered collagen gels containing human aortic smooth muscle cells and human aortic valvular interstitial cells, both isolated from noncalcific heart transplant tissue. The compacted collagen gels were cultured in osteogenic media for 3 weeks in a custom-designed bioreactor and all assessments were performed at the end of the culture period. The major finding of this study is that bone morphogenic protein (BMP)-2 and BMP-4 and transforming growth factor-β1 mRNA expression significantly changed in response to the magnitude of applied strain in valvular cells, while the lowest expression was observed for the representative physiological strain. On the other hand, mRNA expression in vascular cells did not vary in response to the magnitude of strain. Regarding BMP-2 and BMP-4 protein expression determined by immunostaining, trends were similar to mRNA expression in vascular and valvular cells, where only valvular cells showed a varied protein expression depending on the magnitude of the strain applied. Our results suggest that cellular differences exist between vascular and valvular cells in their response to altered levels of cyclic strain during calcification.

Comparison of calcification potential of valvular interstitial cells isolated from individual aortic valve cusps

BACKGROUND Calcific aortic valve disease (CAVD) is one of the most prevalent disorders among the elderly in developed countries. CAVD develops via cell-mediated processes, and clinical data show that CAVD initiates mostly in the noncoronary cusp of the aortic valve. Valvular interstitial cells (VICs) populate the inside of heart valves and are a heterogeneous cell population. The goal of this study is to elucidate the difference in calcification potential among VICs isolated from the left, right, and noncoronary cusps of porcine aortic valves. METHODS AND RESULTS VICs were isolated from each of the aortic valve cusps and cultured in calcifying medium for 14days to induce calcification. The samples were assessed for calcium deposits, nodule formation, and calcific markers using alizarin red and Von Kossa staining, alkaline phosphatase (ALP) staining, ALP enzyme activity assay, and Western blot. Extracellular matrix production and degradation were measured using collagen and glycosaminoglycan (GAG) assay and gelatin zymography. We observed that VICs isolated from the noncoronary cusp expressed greatest amount of the above calcific markers as compared to the coronary cusps. Also, collagen and GAG content was the greatest in noncoronary VICs. However, our zymography results showed significant difference only for active matrix metalloproteinase-2 expression between right and noncoronary VICs. CONCLUSION Our results suggest that VICs among the three cusps within aortic valve might be inherently different, where a subpopulation of VICs might be predisposed to calcification.

Identification of side- and shear-dependent microRNAs regulating porcine aortic valve pathogenesis

Aortic valve (AV) calcification is an inflammation driven process that occurs preferentially in the fibrosa. To explore the underlying mechanisms, we investigated if key microRNAs (miRNA) in the AV are differentially expressed due to disturbed blood flow (oscillatory shear (OS)) experienced by the fibrosa compared to the ventricularis. To identify the miRNAs involved, endothelial-enriched RNA was isolated from either side of healthy porcine AVs for microarray analysis. Validation using qPCR confirmed significantly higher expression of 7 miRNAs (miR-100, -130a, -181a/b, -199a-3p, -199a-5p, and -214) in the fibrosa versus the ventricularis. Upon bioinformatics analysis, miR-214 was selected for further investigation using porcine AV leaflets in an ex vivo shear system. Fibrosa and ventricularis sides were exposed to either oscillatory or unidirectional pulsatile shear for 2 days and 3 & 7 days in regular and osteogenic media, respectively. Higher expression of miR-214, increased thickness of the fibrosa, and calcification was observed when the fibrosa was exposed to OS compared to the ventricularis. Silencing of miR-214 by anti-miR-214 in whole AV leaflets with the fibrosa exposed to OS significantly increased the protein expression of TGFβ1 and moderately increased collagen content but did not affect AV calcification. Thus, miR-214 is identified as a side- and shear-dependent miRNA that regulates key mechanosensitive gene in AV such as TGFβ1.

In‐vitro analysis of early calcification in aortic valvular interstitial cells using Laser‐Induced Breakdown Spectroscopy (LIBS)

Calcific aortic valve disease (CAVD) is a major cardiovascular disorder caused by osteogenic differentiation of valvular interstitial cells (VICs) within aortic valves. Conventional methods like colorimetric assays and histology fail to detect small calcium depositions during in-vitro VIC cultures. Laser-induced breakdown spectroscopy (LIBS) is a robust analytical tool used for inorganic materials characterizations, but relatively new to biomedical applications. We employ LIBS, for the first time, for quantitative in-vitro detection of calcium depositions in VICs at various osteogenic differentiation stages. VICs isolated from porcine aortic valves were cultured in osteogenic media over various days. Colorimetric calcium assays based on arsenazo dye and Von Kossa staining measured the calcium depositions within VICs. Simultaneously, LIBS signatures for Ca I (422.67 nm) atomic emission lines were collected for estimating calcium depositions in lyophilized VIC samples. Our results indicate excellent linear correlation between the calcium assay and our LIBS measurements. Furthermore, unlike the assay results, the LIBS results could resolve calcium signals from cell samples with as early as 2 days of osteogenic culture. Quantitatively, the LIBS measurements establish the limit of detection for calcium content in VICs to be ∼0.17±0.04 μg which indicates a 5-fold improvement over calcium assay. Picture: Quantitative LIBS enables in-vitro analysis for early stage detection of calcium deposition within aortic valvular interstitial cells (VICs).

Design considerations and challenges for mechanical stretch bioreactors in tissue engineering

With the increase in average life expectancy and growing aging population, lack of functional grafts for replacement surgeries has become a severe problem. Engineered tissues are a promising alternative to this problem because they can mimic the physiological function of the native tissues and be cultured on demand. Cyclic stretch is important for developing many engineered tissues such as hearts, heart valves, muscles, and bones. Thus a variety of stretch bioreactors and corresponding scaffolds have been designed and tested to study the underlying mechanism of tissue formation and to optimize the mechanical conditions applied to the engineered tissues. In this review, we look at various designs of stretch bioreactors and common scaffolds and offer insights for future improvements in tissue engineering applications. First, we summarize the requirements and common configuration of stretch bioreactors. Next, we present the features of different actuating and motion transforming systems and their applications. Since most bioreactors must measure detailed distributions of loads and deformations on engineered tissues, techniques with high accuracy, precision, and frequency have been developed. We also cover the key points in designing culture chambers, nutrition exchanging systems, and regimens used for specific tissues. Since scaffolds are essential for providing biophysical microenvironments for residing cells, we discuss materials and technologies used in fabricating scaffolds to mimic anisotropic native tissues, including decellularized tissues, hydrogels, biocompatible polymers, electrospinning, and 3D bioprinting techniques. Finally, we present the potential future directions for improving stretch bioreactors and scaffolds.

Understanding the Role of Sex in Heart Valve and Major Vascular Diseases

Cardiovascular disease (CVD) is the major cause of mortality in the elderly population. The cost of CVD treatment and surgeries was over

A study of extracellular matrix remodeling in aortic heart valves using a novel biaxial stretch bioreactor

300 billion in the United States alone in 2010, making this disorder a critical healthcare issue. Many studies have suggested sex as a risk factor for heart valve and major vascular diseases, such as aortic valve stenosis, mitral prolapse and regurgitation, atherosclerosis, coronary artery disease, and abdominal aortic aneurysm. Unfortunately, only a handful of studies have illustrated the role of sex in the etiology and progression of these disorders. Moreover, knowledge of biomolecular factors that affect these diseases in men and women is very limited. Numerous clinical studies have revealed obvious differences in the prevalence of these diseases between the sexes. These reports were supported by a few molecular and cellular physiology studies that associated this difference to sex and sex hormones. In particular, male sex has commonly been identified as a risk factor for majority of heart valve and vascular diseases, whereas females have been identified as higher risk for certain disorders as well. In addition, menopause is a critical issue that turns the tables against women and enhances complications in their cardiovascular structure due to hormonal change. In this review, major vascular and heart valve diseases for which sex is associated as a risk factor have been reviewed to highlight the importance of this risk factor in CVDs.

Sex-related differences in matrix remodeling and early osteogenic markers in aortic valvular interstitial cells

In aortic valves, biaxial cyclic stretch is known to modulate cell differentiation, extracellular matrix (ECM) synthesis and organization. We designed a novel bioreactor that can apply independent and precise stretch along radial and circumferential directions in a tissue culture environment. While this bioreactor can be used for either native or engineered tissues, this study determined matrix remodeling and strain distribution of aortic cusps after culturing under biaxial stretch for 14 days. The contents of collagen and glycosaminoglycans were determined using standard biochemical assays and compared with fresh controls. Strain fields in static cusps were more uniform than those in stretched cusps, which indicated degradation of the ECM fibers. The glycosaminoglycan content was significantly elevated in the static control as compared to fresh or stretched cusps, but no difference was observed in collagen content among the groups. The strain profile of freshly isolated fibrosa vs. ventricularis and left, right, and noncoronary cusps were also determined by Digital Image Correlation technique. Distinct strain patterns were observed under stretch on fibrosa and ventricularis sides and among the three cusps. This work highlights the critical role of the anisotropic ECM structure for proper functions of native aortic valves and the beneficial effects of biaxial stretch for maintenance of the native ECM structure.