PARACRINE IGF/IGFBP INTERACTIONS IN VIVO

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DESCRIPTION: (Adapted from the applicant's abstract): This is a new R01 grant which focuses on the use of tissue specific transgenic expression to explore the role of IGF-1 and IGFBP-4 and 5 on smooth muscle growth and function in the mouse. In preliminary work, the investigator has expressed IGF-1 and IGFBP-4 using a smooth muscle a-actin promoter (SMP8) which allows for expression in uterus, urinary bladder, intestines, and arteriole. The phenotype of the IGF-1 expression is quite interesting with different patterns of organ remodeling in different tissues. Thus, in bladder and stomach there is concentric thickening of the muscular layer, whereas in intestine and uterus, there is primarily longitudinal growth. Expression of IGFBP-4 has a reciprocal phenotype with hypoplasia of smooth muscle in bladder, stomach, and arteriole wall. The current proposal builds on these preliminary data with four specific aims. Dr. Fagin will determine the impact of IGF-1 over-expression on smooth muscle proliferation, apoptosis and differentiation in vivo using this transgenic model. In addition to examining the morphology of the tissues, rates of cell mitosis (by BUDR labeling) and rates of apoptosis using the TUNEL assay will be measured. He will also examine the effects on contractility and sensitivity to depolarization. Since he has previously demonstrated that the mouse model of IGF-1 over-expression in smooth muscle has circulating levels of IGF in the normal range, this will enable examination of the action of this growth factor at the paracrine level. He will determine the effects of IGF-1 on contractile properties of vascular smooth muscle. This will include studies of calcium flux in muscles, as well a measurements of hemodynamic parameters such as blood pressure, peripheral resistance and pulse rate. Since IGFBP-4 transgenic mice develop moderate degree of smooth muscle hypoplasia, he will determine the role of proteolysis of IGFBP by generating transgenic mice carrying a modified IGFBP-4 which is resistant to protease digestion (through in vitro mutagenesis). He will also determine the interaction between IGF-1 and IGFBP-4 transgenics by co- expression, since there are data to suggest that IGF-1 binding to IGFBP-4 is required for proteolysis. The final specific aim, Dr. Fagin will determine the role of IGFBP-5 using the transgenic approach. In contrast to IGFBP-4, IGFBP-5 is believed to associated with extra-cellular matrix where it is stabilized and protected from proteolysis. It has also been shown to have inhibitory effect on IGF action in several cell types. To test the in vivo physiological role of this protein, the investigator will express IGFBP-5 on his transgenic promoter either in its native form or in a form which has undergone in vitro mutagenesis to modify the site of extracellular matrix binding. He will also study the effects of crossing these mice will the IGF transgenic mice to study the impact of co-expression on phenotype.