Jai Prakash Muyal

Therapeutic Potential of Growth Factors in Pulmonary Emphysematous Condition

Pulmonary emphysema is a major manifestation of chronic obstructive pulmonary disease (COPD), which is characterized by progressive destruction of alveolar parenchyma with persistent inflammation of the small airways. Such destruction in the distal respiratory tract is irreversible and irreparable. All-trans-retinoic acid was suggested as a novel therapy for regeneration of lost alveoli in emphysema. However, profound discrepancies were evident between studies. At present, no effective therapeutic options are available that allow for the regeneration of lost alveoli in emphysematous human lungs. Recently, some reports on rodent’s models have suggested the beneficial effects of various growth factors toward alveolar maintenance and repair processes.

El factor de crecimiento queratinocítico humano recombinante induce la progresión de la supervivencia celular mediada por Akt en ratones enfisematosos

INTRODUCTION Emphysema has been associated with decreased VEGF and VEGFR-2 expression and the presence of high numbers of apoptotic alveolar cells. Keratinocyte growth factor stimulates VEGF synthesis which in turn confers normal lung structure maintenance via the Akt pathway. In this study the potential role of rHuKGF in the improvement of deregulated Akt mediated cell survival pathway in emphysematous mice was investigated. METHODS Three experimental groups, i.e., emphysema, treatment and control groups, were prepared. Lungs of mice were treated on 3 occasions by oropharyngeal instillation of 10mg rHuKGF per kg body weight after induction of emphysema with porcine pancreatic elastase. Subsequently, lung tissues from mice were collected for histopathology and molecular biology studies. RESULTS AND DISCUSSION Histopathology photomicrographs and destructive index analysis have shown that elastase-induced airspace enlargement and loss of alveoli recovered in the treatment group. rHuKGF stimulates VEGF production which in turn induces the Akt mediated cell survival pathway in emphysematous lungs. mRNA expression of VEGF, VEGFR, PI3K and Akt was significantly increased while Pten, Caspase-9 and Bad was notably decreased in treatment group when compared with emphysema group, being comparable with the control group. Moreover, VEGF protein expression was in accordance with that found for mRNA. CONCLUSION Therapeutic rHuKGF supplementation improves the deregulated Akt pathway in emphysema, resulting in alveolar cell survival through activation of the endogenous VEGF-dependent cell survival pathway. Hence rHuKGF may prove to be a potential drug in the treatment of emphysema.

DNA-Microarray Technology: Comparison of Methodological Factors of Recent Technique Towards Gene Expression Profiling

DNA-microarray technology is a powerful tool to explore the potentials of functional genomics. Almost a decade back, it was not so easy to trace the molecular pathway process in the pathogenesis of diseases. Today DNA-microarray technology allows identifying candidate genes that are implicated in the development and progression of diseases. Because this technology is new, it remains a challenge to perform DNA-microarray experiments in the laboratory. All the technical factors associated with DNA-microarray experiments have a strong impact on the results and therefore, all parts/steps of the protocol, in particular sample preparation (i.e., RNA isolation, RNA preamplification, and cDNA labeling), hybridization, washing, and scanning settings need to be checked and optimized to obtain reliable results from DNA-microarray experiments.

Effect of recombinant human keratinocyte growth factor in inducing Ras–Raf–Erk pathway-mediated cell proliferation in emphysematous mice lung

Abstract Context: Pulmonary emphysema is resulted due to destruction of the structure of the alveoli. Recently, exogenous recombinant human Keratinocyte growth factor (rHuKGF) has been reported to induce the regeneration of gas exchange structures. However, the molecular mechanisms governing this process are so far unknown. Objective: The objective of this study was to investigate the effect of rHuKGF in the lungs of emphysema-challenged mice on Ras–Raf–Erk (Erk, extracellular signal-regulated kinase) mediated signaling pathway that regulates alveolar epithelial cell proliferation. Methods: Three experimental groups (i.e. emphysema, therapy and control group) were prepared. Lungs of mice were therapeutically treated at three occasions by oropharyngeal instillation of 10 mg rHuKGF per kg body weight after induction of emphysema by porcine pancreatic elastase (PPE). Subsequently, lung tissues from each mouse were collected for histopathology and molecular biology studies. Results and discussion: Histopathology photomicrographs and Destructive Index analysis have shown that elastase induced airspace enlargement and loss of alveoli were recovered in therapy group. Moreover, proliferating cell nuclear antigen (PCNA) at mRNA and protein expression level was markedly increased in therapy group than emphysema group. Upon validation at mRNA level, expressions of FGF-7, FGF-R, Ras, c-Raf, Erk-1, Erk-2, c-Myc and were significantly increased, whereas Elk-1 was notably decreased in therapy group when compared with emphysema group and were well comparable with the control group. Conclusion: Therapeutic supplementation of rHuKGF rectifies the deregulated Ras–Raf–Erk pathway in emphysema condition, resulting in alveolar epithelium regeneration. Hence, rHuKGF may prove to be a potential drug in the treatment of emphysema.

Recombinant human keratinocyte growth factor attenuates apoptosis in elastase induced emphysematous mice lungs

Abstract Alveolar cell apoptosis is one of the potential factors involved in the pathogenesis of emphysema. Recently, exogenous recombinant human keratinocyte growth factor (rHuKGF) has been reported to induce the regeneration of gas exchange structures. Therefore, the rationale of the present study was to investigate the potential effect of rHuKGF in ameliorating tissue destruction in the emphysematous mice lungs. Four experimental groups (i.e. control-, emphysema-, therapy- and therapy control-group) were prepared. Subsequently, lungs from each mouse were collected for comet assay, elastase activity assay, antioxidant activity assay and real-time PCR based analyses. Comet assay analysis demonstrated the reduced tail DNA % and olive tail moment in therapy group. rHuKGF supplementation in emphysematous mice caused a significant reduction in the elastase activity levels along with reduction in activity of CAT, SOD and GPx. Furthermore, based on mRNA expression studies, the supplementation of rHuKGF ameliorated the induced apoptosis pathway in emphysematous mice lungs. Moreover, due to rHuKGF supplementation, TNF-α and p53 expression and production were markedly decreased in emphysematous mice lungs. Thus, therapeutic supplementation of rHuKGF might have reversed the alveolar cell loss in elastase induced emphysematous mice lungs by reducing DNA damage and maintaining antioxidant activities.

ATRA reduces inflammation and improves alveolar epithelium regeneration in emphysematous rat lung

INTRODUCTION Pulmonary emphysema characterized by alveolar wall destruction is resultant of persistent chronic inflammation. All-trans retinoic acid (ATRA) has been reported to reverse elastase-induced emphysema in rats. However, the underlying molecular mechanisms are so far unknown. OBJECTIVE To investigate the therapeutic potential effect of ATRA via the amelioration of the ERK/JAK-STAT pathways in the lungs of emphysematous rats. METHODS In silico analysis was done to find the binding efficiency of ATRA with receptor and ligands of ERK & JAK-STAT pathway. Emphysema was induced by porcine pancreatic elastase in Sprague-Dawley rats and ATRA was supplemented as therapy. Lungs were harvested for histopathological, genomics and proteomics analysis. RESULTS AND DISCUSSION In silico docking, analysis confirms that ATRA interferes with the normal binding of ligands (TNF-α, IL6ST) and receptors (TNFR1, IL6) of ERK/JAK-STAT pathways respectively. ATRA restored the histology, proteases/antiproteases balance, levels of inflammatory markers, antioxidants, expression of candidate genes of ERK and JAK-STAT pathways in the therapy group. CONCLUSION ATRA ameliorates ERK/JAK-STAT pathway in emphysema condition, resulting in alveolar epithelium regeneration. Hence, ATRA may prove to be a potential drug in the treatment of emphysema.

rHuKGF ameliorates protease/anti-protease imbalance in emphysematous mice

We attempted to elucidate the beneficial role of rHuKGF supplementation in the amelioration of protease/antiprotease imbalance and TGF-β1 signaling pathway leading to alveolar tissue maintenance in elastase induced emphysematous mice. Thirty two male C57BL mice were divided into four groups i.e. control, emphysema, therapy and rHuKGF only and were oropharyngeally instilled with saline/porcine pancreatic elastase/rHuKGF. Subsequently, lungs from mice were collected for histopathology and molecular biology studies. rHuKGF supplementation significantly ameliorated the mRNA expressions of CRP, TNF-α, MMP-2, MMP-7, MMP-8, MMP-9, MMP-12, A1AT, TIMP1, TIMP2, PCNA, Ki67, SPB, SPC and PdPn. MMP-2 and TIMP-1 enzyme activity was resolved due to rHuKGF. Likewise, due to rHuKGF supplementation the protein expressions of CRP, MMP2, MMP7, MMP8 & CTSE, SERPINE1, SERPINA1, TIMP4, GSTA1, HDAC3, PCNA, CDH1, SP-B & SP-C were ameliorated. Moreover, the mRNA expressions of overall TGFβ-1 pathway was also significantly ameliorated due to rHuKGF supplementation. Lung histopathology represents recovery of lost alveolar septa due to rHuKGF supplementation. Moreover, positive DAB staining of PCNA, SP-B & SP-C was observed due to rHuKGF supplementation at tissue level. rHuKGF is therapeutically potent in maintaining pulmonary tissue integrity by amelioration of protease/antiprotease imbalance and TGFβ-1 pathway in emphysema.