No abstract

Vascular Endothelial Growth Factor (VEGF) has a potent role in tumorigenesis and metastasis. VEGF gene is highly polymorphic. Specific variants have been associated with risk and disease progression in disorders with microvascular involvement such as diabetes mellitus and cancer. However, no study has reported association analysis of common single nucleotide variants (SNVs) in the promoter and 3' untranslated region (3'-UTR) with head and neck cancers (HNCs). The present study addresses this gap. It investigates the association of two SNVs, -2578 C/A (rs699947) in the promoter region and +936 C/T (rs3025039) in 3'-UTR of the VEGF gene, with risk of HNCs and tumour characteristics. After the ethical approval and informed consent, 323 participants were enrolled in the study. In the case-control component of the study, there were 121 HNC patients and 202 controls. Demographic details and medical history were recorded. Peripheral blood samples (10ml) were collected in ACD-coated vacutainers. DNA isolation was carried out by organic-solvent method. PCR-RFLP methods were optimized for the genotyping of selected SNVs. The protocol was validated by Sanger sequencing. The susceptibility association was determined by using genetic models applying Hardy-Weinberg equilibrium. Statistical analyses included Chi-squared test of independence or Fisher's exact test with significance at p-value <0.05, and odds ratios with 95% confidence interval. VEGF -2578 A-allele, A-carrier genotype (dominant model), and AA genotype (co-dominant model) were significantly associated with protective effect against HNCs (p-values <0.01, <0.042, and < 0.009, respectively). The odds ratios (95% CI) were: 0.651 (0.469 - 0.904), 0.613 (0.381 - 0.985), and 0.393 (0.193 - 0.804), respectively. The association analysis of VEGF +936 C/T polymorphism showed that T-allele, CT (over-dominant model), and T-carrier (dominant model) genotypes were significantly associated with the risk for head and neck cancers (p-values < 0.047, < 0.037, and < 0.038, respectively). The odds ratios (95% CI) were 1.882 (1.001 - 3.536); 2.060 (1.035 - 4.102); and 2.023 (1.032 - 3.966), respectively. Additionally, VEGF +936 CT and T-carrier genotypes showed significant associations with higher tumour grade (p-value <0.029, and <0.037). The odds ratios (95% CI) were 10.00 (1.435-72.806) and 9.00 (1.294 - 62.594), respectively. VEGF -2578 C/A and +936 C/T may be further investigated as susceptibility genetic markers for HNCs. Specifically, -2578 A-variant has a protective effect, whereas +936 T-variant increases the risk. Furthermore, +936 CT and T-carrier genotypes are also associated with higher tumour grade. The present study is the first positive association analysis report of both selected SNVs with HNCs in any population.

Direct reprogramming of autologous somatic cells into cardiomyocytes is a novel approach which is being employed for cardiac regeneration. Multiple studies have combined different cardiac-specific factors that could directly reprogram cardiac fibroblasts into induced cardiomyocyte-like cells. However, this approach needs heart biopsy for the isolation of autologous fibroblasts which involves extensive surgical procedures that require precision. In this study, we aimed to develop an approach which can directly reprogram somatic cells into cardiomyocyte-like cells using a combination of pluripotency and cardiac transcription factors. Skin fibroblasts were isolated from rat neonatal pups and were induced into cardiomyocyte-like cells using non-viral integration of cardiac transcription factors (GATA4, Mef2c and Nkx2.5) and OKSIM plasmid carrying the iPSC factors, Sox2, Oct4, Klf4 and cMyc. After 72 h, cells were analyzed for the expression of cardiac markers by qPCR and immunocytochemistry. Gene expression analysis showed significantly higher expression of cardiac markers GATA4, cMHC, Mef2c, cTnT, cTnI, and Nkx2.5 Immunostaining confirmed the expression of cardiac proteins GATA4, cMHC, cTnT, cTnI, and Nkx2.5. These results imply that the transfected cells started differentiating towards cardiac lineage. Transfected cells were also transplanted in rat myocardial infarction (MI) model immediately after ligation of left ventricle descending (LAD) artery. After 2 and 4 weeks of cell transplantation, the animals were assessed for cardiac function via echocardiography. They showed significant improvement in the cardiac function as compared to MI and non-transfected groups. After 4 weeks, the hearts were harvested, and histological analysis was performed for the assessment of fibrosis and left ventricular wall thickness. Rats transplanted with transfected cells showed significantly reduced fibrosis and increased wall thickness as compared to MI and non-transfected groups. In conclusion, transient and combined expression of cardiac transcription and iPSC factors in neonatal somatic cells leads to the transdifferentiation of skin cells into myogenic lineage. This approach can be useful for future therapeutic application for cardiovascular diseases.