Methods for diagnosing pulmonary hypertension

Abstract

Hypoxia induced mitogenic factor (HIMF) is a member of the “found in inflammatory zone” (FIZZ)/resistin family of proteins and has potent mitogenic, angiogenic, and vasoconstrictive effects in the lung vasculature. The receptor/binding partners for this family of proteins have been largely unknown. We identified Bruton's tyrosine kinase (BTK) as a functional HIMF binding partner through GST-HIMF pull-downs and mass spectrometry. Using primary cultured HIMF-stimulated murine bone marrow cells, we demonstrated that BTK was recruited to the leading edge of the cells. We also demonstrated that BTK and the closely related tyrosine kinase Fyn, colocalized at the growth cone process in these cells. HIMF stimulation induced BTK autophosphorylation, which peaked at 2.5 minutes. A transwell migration assay showed that treatment with recombinant murine HIMF induced migration of primary cultured bone marrow cells, which was completely blocked by the BTK inhibitor, LFM-A13. In vivo studies, using the rat hindlimb ischemia model, revealed that HIMF can stimulate angiogenesis in the hypoxic tissue probably through inducing the migration of endothelial progenitor cells (EPCs) to areas of active angiogenesis. Our results indicate that HIMF may acts as a chemotactic molecule in stimulating the migration of leukocytes/EPCs from bone marrow to targeted tissues through activation of the BTK pathway.

1 - 23 . (canceled) 
     
     
         24 . A method to diagnose pulmonary hypertension in a subject, comprising:
 (a) determining the amount of HIMF (SEQ ID NO: 1 or 2) in a serum sample of a subject using an enzyme-linked immunosorbent assay that comprises an antibody that specifically binds Resistin or RELMbeta; and   (b) diagnosing pulmonary hypertension in the subject based on elevated levels of HIMF as compared to normal control subjects who do not have pulmonary hypertension.   
     
     
         25 . The method of claim  1 , further comprising the step of administering to the subject an antibody which specifically binds to HIMF (SEQ ID NO: 1 or 2).