Research Projects

Neutrophil extracellular traps (NETs) were found to be a new perpetrator in the progression of non-ischemic cardiac hypertrophy. Studies have shown that deletion of the extra domain A fibronectin (EDA-FN) exon or antibodies against EDA-FN protected the mice against neutrophil infiltration and NETs formation following stroke and myocardial ischemia-reperfusion injury. Of note, EDA-FN is normally not present in the circulation or healthy arteries of both rodents and humans; however, it is specifically upregulated in patients with diabetes, vascular hypertension, atherosclerosis, thrombosis, myocardial ischemia-reperfusion injury and stroke. Inhibition of EDA-FN significantly protected the rodents from insulin resistance, atherosclerosis, arterial thrombosis, myocardial ischemia-reperfusion injury and ischemic stroke through toll-like receptor 4 (TLR4). We found isoproterenol (ISO) infusion for two weeks increased heart weight to body weight or tibia weight ratio. Immunohistochemistry (IHC) confirmed severe cardiac hypertrophy as we found positive for alpha-smooth muscle actin, periostin and collagen. Hematoxylin and eosin staining showed massive leukocyte infiltration. IHC and Western blot data have shown neutrophil elastase expression, indicating neutrophil infiltration. Comorbid hypercholesteremia further aggravated the cardiac hypertrophic phenotype and, significantly decreased preload-recruitable stroke work and declined the end-systolic pressure-volume relationship slope. The tissue was positive for EDA-FN and TLR4 and its inhibition protected the rat against cardiac hypertrophy by significantly decreasing the heart weight to tibia weight ratio. Co-immunoprecipitation data have shown a physical interaction between TLR4 and neutrophil elastase, a serine protease loaded on NETs. We propose to test the role of EDA-FN and TLR4 axis in neutrophil infiltration and NETosis in non-ischemic cardiac hypertrophy. Neutrophils, T-cell receptors and B-lymphocytes infiltration in heart tissue will be quantified by using flow cytometry. Immunofluorescence staining will be done to identify and quantify NETs by probing for citrullinated histone H3 and neutrophil elastase in the heart tissue. Antibodies against EDA-FN and inhibitors against TLR4 will be given to protect the rats against neutrophil infiltration and NETs formation. Adenoviral-mediated, EDA-FN will be transiently overexpressed in vivo to unravel its role in neutrophil infiltration and NETs formation via TLR4. Furthermore, pressure-volume loop analysis will be done to measure heart function following the intervention. EDA-FN get expressed in disease conditions and binds to TLR4 independent of its lipopolysaccharide-protein binding region. Thus can be considered as a potential therapeutic targeting against neutrophil infiltration and NETs formation devoid of side effects.