Subtype and Lineage-Mediated Protocol for Standardizing Activin/Nodal and BMP Signaling for hiPSC-Derived Cardiomyocyte Differentiation.

The adept and systematic differentiation of embryonic stem cells (ESCs) and human-induced pluripotent stem cells (hiPSCs) to diverse lineage-prone cell types involves crucial step-by-step process that mimics the vital strategic commitment phase that is usually observed during the process of embryo development. The development of precise tissue-specific cell types from these stem cells indeed plays an important role in the advancement of imminent stem cell-based therapeutic strategies. Therefore, the usage of hiPSC-derived cell types for subsequent cardiovascular disease modeling, drug screening, and therapeutic drug development undeniably entails an in-depth understanding of each and every step to proficiently stimulate these stem cells into desired cardiomyogenic lineage. Thus, to accomplish this definitive and decisive fate, it is essential to efficiently induce the mesoderm or pre-cardiac mesoderm, succeeded by the division of cells into cardiovascular and ultimately ensuing with the cardiomyogenic lineage outcome. This usually commences from the earliest phases of pluripotent cell induction. In this chapter, we discuss our robust and reproducible step-wise protocol that will describe the subtype controlled, precise lineage targeted standardization of activin/nodal, and BMP signaling molecules/cytokines, for the efficient differentiation of ventricular cardiomyocytes from hiPSCs via the embryoid body method. In addition, we also describe techniques to dissociate hiPSCs, hiPSC-derived early cardiomyocytes for mesoderm and pre-cardiac mesoderm assessment, and hiPSC-derived cardiomyocytes for early and mature markers assessment.

The Role of Bone Morphogenetic Protein Receptor Type 2 (BMPR2) and the Prospects of Utilizing Induced Pluripotent Stem Cells (iPSCs) in Pulmonary Arterial Hypertension Disease Modeling.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by increased pulmonary vascular resistance (PVR), causing right ventricular hypertrophy and ultimately death from right heart failure. Heterozygous mutations in the bone morphogenetic protein receptor type 2 (BMPR2) are linked to approximately 80% of hereditary, and 20% of idiopathic PAH cases, respectively. While patients carrying a BMPR2 gene mutation are more prone to develop PAH than non-carriers, only 20% will develop the disease, whereas the majority will remain asymptomatic. PAH is characterized by extreme vascular remodeling that causes pulmonary arterial endothelial cell (PAEC) dysfunction, impaired apoptosis, and uncontrolled proliferation of the pulmonary arterial smooth muscle cells (PASMCs). To date, progress in understanding the pathophysiology of PAH has been hampered by limited access to human tissue samples and inadequacy of animal models to accurately mimic the pathogenesis of human disease. Along with the advent of induced pluripotent stem cell (iPSC) technology, there has been an increasing interest in using this tool to develop patient-specific cellular models that precisely replicate the pathogenesis of PAH. In this review, we summarize the currently available approaches in iPSC-based PAH disease modeling and explore how this technology could be harnessed for drug discovery and to widen our understanding of the pathophysiology of PAH.

Implication of Lipids in Calcified Aortic Valve Pathogenesis: Why Did Statins Fail?

Calcific Aortic Valve Disease (CAVD) is a fibrocalcific disease. Lipoproteins and oxidized phospholipids play a substantial role in CAVD; the level of Lp(a) has been shown to accelerate the progression of valve calcification. Indeed, oxidized phospholipids carried by Lp(a) into the aortic valve stimulate endothelial dysfunction and promote inflammation. Inflammation and growth factors actively promote the synthesis of the extracellular matrix (ECM) and trigger an osteogenic program. The accumulation of ECM proteins promotes lipid adhesion to valve tissue, which could initiate the osteogenic program in interstitial valve cells. Statin treatment has been shown to have the ability to diminish the death rate in subjects with atherosclerotic impediments by decreasing the serum LDL cholesterol levels. However, the use of HMG-CoA inhibitors (statins) as cholesterol-lowering therapy did not significantly reduce the progression or the severity of aortic valve calcification. However, new clinical trials targeting Lp(a) or PCSK9 are showing promising results in reducing the severity of aortic stenosis. In this review, we discuss the implication of lipids in aortic valve calcification and the current findings on the effect of lipid-lowering therapy in aortic stenosis.

Functional characterization of promoter region polymorphisms of human CYP2C19 gene.

CYP2C19 is an enzyme involved in the metabolism of several clinically important drugs. The variations in the CYP2C19 promoter region may alter the transcription of the gene by altering the interaction between the trans and cis-acting elements. In the present study, CYP2C19 promoter region with different variant alleles were cloned into a pGL-3 basic luciferase reporter vector and transfected into HepG2 cell lines. Subsequently, dual luciferase activity was measured to evaluate the activity of the promoter region. Gel shift assays with predicted binding sites for CCAAT displacement protein, activating transcription factor-2 and glucocorticoid receptor were performed. Results from this study indicate that few variations present in the putative cis-acting elements of the CYP2C19 promoter region such as -1442T>C, -779A>C and -98T>C -1498T>G and -828del>T alter the transcription of the gene. Specific binding with nuclear proteins was also observed in gel shift assays. This may account for the interindividual variations in gene expression and genotype dependant differences in gene transcription. The results also suggest the role of activating transcription factor-2 and CCAAT displacement repressor protein on CYP2C19 gene transcription.

Allele, Genotype and Haplotype Structures of Functional Polymorphic Variants in Endothelial Nitric Oxide Synthase (eNOS), Angiotensinogen (ACE) and Aldosterone Synthase (CYP11B2) Genes in Healthy Pregnant Women of Indian Ethnicity.

BACKGROUND: Variants in the candidate genes eNOS, CYP11B2 and ACE have been implicated as liable biomarkers that can predict complications like hypertension and preeclampsia. Studies on the impact and distribution of these variants on healthy pregnancy have not been done so far in south Indian or in any of the native Indian population. Examining these variants could lay a strong basis in understanding the genetic aspects of preeclampsia and further offer effective means in early risk assessment in a preeclampsia. METHODS: Genotyping for 303 unrelated healthy women of Tamilian origin who underwent uncomplicated term pregnancies was executed by PCR-RFLP for eNOS, CYP11B2 and ACE variants. Haplotype assessment and pairwise linkage disequilibrium (LD) investigation were performed by Haploview software. RESULTS: The prevalence of eNOS variants (-786T>C, Glu298Asp and intron 4 VNTR) was 12%, 21.6% and 21.1%, respectively. The incidence of CYP11B2 (-344 C>T) and ACE (287 bp Alu I/D) variants was found to be 43.8% and 42.7%. The observed frequencies of the studied polymorphisms did not diverge from the HWE (p>0.05). Significant LD was observed between 3 eNOS gene polymorphisms. Six different haplotype structures with a frequency of >1% were generated from three eNOS variants. Among the haplotypes generated, the haplotype T-4b-G was the most common with the frequency of 64.4%. There was a statistically significant inconsistency in the study population in comparison to other global races. CONCLUSION: The outcome of this study could be used for investigating future therapeutic value of the variants in a preeclamptic set-up which could pose a credible diagnostic potential for primary risk assessment of women susceptible to preeclampsia/other pregnancy related complications.

Relative Copy Number Variations of CYP2C19 in South Indian Population.

CYP2C19 is a polymorphic enzyme involved in the metabolism of clinically important drugs. Genotype-phenotype association studies of CYP2C19 have reported wide ranges in the metabolic ratios of its substrates. These discrepancies could be attributed to the variations in the promoter region and this aspect has been reported recently. The observations in the recent reports on the influence of promoter region variants on the metabolism of CYP2C19 substrates might also have been influenced by the copy number variations of CYP2C19. In this paper, we describe copy number variations of CYP2C19 using real-time polymerase chain reaction by comparative Ct method. No copy number variations were observed in the south Indian population indicating the observed discrepancies in genotype-phenotype association studies might be due to the regulatory region polymorphisms as reported earlier.

Genetic variations and haplotypes of the 5' regulatory region of CYP2C19 in South Indian population.

CYP2C19 is expressed polymorphically with about 21 variant alleles. Genotype-phenotype association studies of CYP2C19 have shown marked deviations, suggesting the presence of other variations in the intronic and 5' regulatory region affecting its expression. This study aims to identify the genetic polymorphisms and construction of haplotypes of variations in 5' regulatory region of CYP2C19 among the South Indian population. CYP2C19 5' regulatory region was amplified and sequenced from the DNA of 58 healthy volunteers of South Indian origin. Genetic analysis revealed the existence of 14 variations including eight novel ones in the 5' regulatory region. Identified novel variations and their percentage frequencies were: -779A>C (16.4), -828T>A (2.6), -934del>T (3.5), -1051T>C (1.72), -1289T>G (3.4), -1442T>C (12.1), -1498T>G (25.0) and -1558T>G (2.6). The reported variations found in the study population and their frequencies were: -98T>C (28.4), -806C>T (2.6), -833del>T (9.5), 889T>G (10.3), -1041A>G (100.0) and -1418C>T (1.7). The two known non synonymous single nucleotide polymorphisms, 681G>A ((*)2 allele) and 636G>A ((*)3 allele) were detected at 0.371 and 0.025 frequencies, respectively. Forty three haplotypes were constructed and linkage disequilibrium analysis showed strong linkage between several variations identified in the gene. Fourteen polymorphisms including 8 novel ones in CYP2C19 5' flanking region are reported for the first time in an Indian population from South India. Results from this study provide additional information for genotyping of CYP2C19 in the South Indian population and probably in the Indian population.

Influence of the genetic polymorphisms in the 5' flanking and exonic regions of CYP2C19 on proguanil oxidation.

CYP2C19 is a polymorphic enzyme which metabolizes several clinically important drugs including proguanil. Variation in the 5' regulatory region may influence CYP2C19 activity. This study evaluates the relationship between proguanil metabolic ratio and genetic variations of CYP2C19 in a South Indian population. Fifty unrelated healthy subjects were genotyped for CYP2C19 (*)2 and (*)3 alleles and the 5' flanking region of CYP2C19 was sequenced. Plasma concentrations of proguanil and cycloguanil were estimated by reverse phase HPLC after single oral doses (200 mg) of proguanil. In silico docking analysis of transcription factors binding to its sites in CYP2C19 5' regulatory region was performed. The mean metabolic ratios (proguanil/cycloguanil) were highest in (*)1/(*)2 or (*)1/(*)3 subjects and in (*)2/(*)2 or (*)2/(*)3 as compared to (*)1/(*)1 subjects. Subjects with promoter region variation -98T>C showed decrease in the metabolic ratios irrespective of other variation, which may explain the deviation from the genotype-phenotype association of CYP2C19. In silico analysis predicted alteration in the interaction of transcription factors to their binding sites in the presence of variant alleles. The results of this study would be useful in predicting interindividual differences in the metabolism of substrates of CYP2C19.