Characterizing Glial Pathways that Promote Brain Vascular Development and Physiology:
The various organs and tissues of the body harbor elaborate networks of blood vessels that display a wide variety of structural and functional features. This vascular heterogeneity is determined, in part, by cues within the local environment. How the vascular endothelium in different organs properly deciphers and integrates these various cues to control cell growth, sprouting and differentiation remains uncharacterized. A significant challenge in vascular biology, therefore, is to identify genes and pathways that determine organ-specific blood vessel heterogeneity. The McCarty lab studies this fundamental problem within the context of the brain, which contains an intricate web of blood vessels that display many unique structural and functional features, including a blood brain barrier (BBB). The BBB regulates the exchange of essential nutrients between the circulation and the neural microenvironment to meet the high metabolic demands of the brain. In the brain, astroglial cells communicate with endothelial cells to control BBB functions via secreted growth factors and ECM proteins. Integrins are receptors for many ECM protein ligands, and integrin-mediated adhesion and signaling events are essential for vascular development and homeostasis in the brain. For example, Dr. McCarty’s group has demonstrated that the glial expressed integrin αvβ8 and its ECM protein ligands, the latent TGFβs, are essential for blood vessel morphogenesis in the embryonic brain. TGFβs are produced by cells as ECM-bound latent complexes, and biochemical studies have shown that αvβ8 integrin binds to RGD peptide sequences within latent TGFβ1 and TGFβ3, mediating TGFβ release from the ECM and receptor engagement. Data from the McCarty lab also reveal that ablation of TGFβ receptor type 2 (Tgfbr2) gene expression in endothelial cells, but not glial cells, results in cerebral vascular pathologies that are identical to those that develop in αvβ8 integrin and TGFβ1/3 mutant mice. We have also found that the endothelial cell-expressed protein neuropilin 1 (Nrp1) acts as a negative regulator of integrin-mediated TGFβ activation and signaling. We are currently investigating additional pathways that control neurovascular development as well as determining how they may be interconnected with αvβ8 integrin and TGFβ signaling events.
Characterizing Astrocyte-Derived Factors that Regulate BBB Development and Pathophysiology:
Perivascular astrocytes intimately associate with endothelial cells and their shared basement membranes within multicellular complexes, or neurovascular units, to regulate the development and homeostasis of the BBB. Signals from perivascular astrocyte end feet promote the formation and maintenance of endothelial cell tight junctions and control expression of important BBB transporter proteins. We have developed a knock-in mouse model in which GFP expression is expressed exclusively in perivascular astrocytes and not in neurons, endothelial cells, pericytes or other glial cell types. We are currently fractionating perivascular astrocytes from these mice and performing whole transcriptome sequencing to identify factors that regulate BBB functions. This mouse model will be a powerful tool for understanding the genetic and biochemical profiles of perivascular astrocytes and their contributions to BBB development and homeostasis in development and cancer. The long-term goal is to therapeutically target the BBB to improve drug delivery to improve the lives of patients with brain cancer and other neurological diseases.
Analyzing Adhesion and Signaling Pathways that Drive Brain Cancer Initiation and Progression:
Glioblastoma (GBM) is a heterogeneous brain cancer that contains sub-populations of highly invasive cells that drive tumor growth, progression and recurrence after therapy. These invasive GBM cells escape surgical resection and remain largely resistant to chemotherapy. The signaling pathways that selectively regulate GBM cell invasive growth remain poorly understood. We made the major discovery that αvβ8 integrin promotes the activation of latent-TGFβs to drive perivascular brain tumor cell invasion. αvβ8 integrin-activated TGFβ signaling is negatively regulated by Nrp1 to control developmental brain angiogenesis, and these pathways are reactivated during GBM progression. The β8 integrin cytoplasmic domain is divergent from other β subunits and lacks conserved peptide motifs, e.g., NPXY, that have well-characterized roles in integrin activation and signaling. Therefore, we performed genetic and biochemical screens to identify factors that bind to the β8 integrin cytoplasmic tail to regulate GBM cell growth or invasion. These efforts led to the discovery of the Rho GTPase effector protein RhoGDI1, the cytoskeletal adaptor proteins Band 4.1B and Spinophilin, and the cytosolic tyrosine phosphatase PTPN12/PTP-PEST. We have also discovered an integrin-regulated protein complex comprised of PTPN12/PTP-PEST and the ubiquitin-dependent segregase valosin-containing protein (Vcp/p97). Using primary patient cells and pre-clinical mouse models of brain cancer, we have discovered novel functions for PTP-PEST and Vcp in regulating GBM cell invasive growth during tumor initiation and progression. We are currently analyzing functions for the PTP-PEST/Vcp complex in promoting the phosphorylation-dependent ubiquitination and degradation of key focal adhesion protein substrates such as p130Cas.
Targeting VEGF-Independent Angiogenic Signaling Pathways in the Brain Cancer Microenvironment:
Growth and progression of primary brain cancer is tightly coupled to the vasculature, which has made GBM an attractive target for anti-angiogenic therapies. However, many targeted agents, including the vascular endothelial growth factor (VEGF) neutralizing antibody Bevacizumab/Avastin, have yielded disappointing results in clinical trials. While many patients display short-term improvement due to blood vessel regression and diminished edema, they invariably relapse due to acquired resistance and the activation of VEGF-independent angiogenic signaling pathways. Hence, it is important to characterize alternative signaling pathways in endothelial cells to identify potential new targets for anti-angiogenic therapies in patients with GBM. Open source genomic databases have been searched to identify genes that are (i) enriched in cerebral endothelial cells during development, (ii) down-regulated in quiescent endothelial cells in the post-natal brain, and (iii) reactivated in angiogenic endothelial cells in malignant brain tumors. These efforts have led to new projects involving novel genes in brain cancer angiogenesis. We are using cutting-edge cell biology techniques, state-of-the-art mouse models and primary patient samples to characterize these genes and pathways that promote angiogenesis and BBB breakdown in GBM.