Craig Crowley

Mice lacking nerve growth factor display perinatal loss of sensory and sympathetic neurons yet develop basal forebrain cholinergic neurons

Homologous recombination was utilized to generate mice with a deletion in the coding sequence of the nerve growth factor (NGF) gene. Animals homozygous for NGF disruption failed to respond to noxious mechanical stimuli, and histological analysis revealed profound cell loss in both sensory and sympathetic ganglia. Within dorsal root ganglia, effects of the mutation appeared to be restricted to small and medium peptidergic neurons. These observations confirm the critical dependence of sensory and sympathetic neurons on NGF and demonstrate that other neurotrophins are not able to compensate for the loss of NGF action on these cells. Examination of the central nervous system revealed that, in marked contrast with neurons of sensory and sympathetic ganglia, basal forebrain cholinergic neurons differentiate and continue to express phenotypic markers for the life span of the null mutant mice. Thus, differentiation and initial survival of central NGF-responsive neurons can occur in the absence of NGF.

Requirements for Binding and Signaling of the Kinase Domain Receptor for Vascular Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is a dimeric hormone that controls much of vascular development through binding and activation of its kinase domain receptor (KDR). We produced analogs of VEGF that show it has two receptor-binding sites which are located near the poles of the dimer and straddle the interface between subunits. Deletion experiments in KDR indicate that of the seven IgG-like domains in the extracellular domain, only domains 2–3 are needed for tight binding of VEGF. Monomeric forms of the extracellular domain of KDR bind ∼100 times weaker than dimeric forms showing a strong avidity component for binding of VEGF to predimerized forms of the receptor. Based upon these structure-function studies and a mechanism in which receptor dimerization is critical for signaling, we constructed a receptor antagonist in the form of a heterodimer of VEGF that contained one functional and one non-functional site. These studies establish a functional foundation for the design of VEGF analogs, mimics, and antagonists.

EphB2 as a Therapeutic Antibody Drug Target for the Treatment of Colorectal Cancer

Analysis of human colorectal cancer specimens revealed overexpression of the EphB2 receptor tyrosine kinase. Monoclonal antibodies (MAbs) to extracellular sequence of EphB2 were raised and tested for activity against colorectal cancer cells. One of the MAbs, 2H9, effectively blocked the interaction of ephB2 with ephrin ligands and inhibited the resulting autophosphorylation of the receptor. However, this antibody did not affect the proliferation of cancer cells expressing ephB2. Immunocytochemical analysis revealed rapid internalization of the MAb 2H9 on binding ephB2, suggesting that target-dependent cell killing could be achieved with an antibody-drug conjugate. When MAb 2H9 was conjugated to monomethylauristatin E through a cathepsin B-cleavable linker, it specifically killed ephB2-expressing cancer cells in vitro and in vivo. Our results suggest that ephB2 is an attractive target for immunoconjugate cancer therapy.

WISP-1 Is an Osteoblastic Regulator Expressed During Skeletal Development and Fracture Repair

Wnt-1-induced secreted protein 1 (WISP-1) is a member of the CCN (connective tissue growth factor, Cyr61, NOV) family of growth factors. Experimental evidence suggests that CCN family members are involved in skeletogenesis and bone healing. To investigate the role of WISP-1 in osteogenic processes, we characterized its tissue and cellular expression and evaluated its activity in osteoblastic and chondrocytic cell culture models. During embryonic development, WISP-1 expression was restricted to osteoblasts and to osteoblastic progenitor cells of the perichondral mesenchyme. In vitro, we showed that WISP-1 expression in differentiating osteoblasts promotes BMP-2-induced osteoblastic differentiation. Using in situ and cell binding analysis, we demonstrated WISP-1 interaction with perichondral mesenchyme and undifferentiated chondrocytes. We evaluated the effect of WISP-1 on chondrocytes by generating stably transfected mouse chondrocytic cell lines. In these cells, WISP-1 increased proliferation and saturation density but repressed chondrocytic differentiation. Because of the similarity between skeletogenesis and bone healing, we also analyzed WISP-1 spatiotemporal expression in a fracture repair model. We found that WISP-1 expression recapitulates the pattern observed during skeletal development. Our data demonstrate that WISP-1 is an osteogenic potentiating factor promoting mesenchymal cell proliferation and osteoblastic differentiation while repressing chondrocytic differentiation. Therefore, we propose that WISP-1 plays an important regulatory role during bone development and fracture repair.