William Schubert

Microvascular Hyperpermeability in Caveolin-1 (−/−) Knock-out Mice TREATMENT WITH A SPECIFIC NITRIC-OXIDE SYNTHASE INHIBITOR,l-NAME, RESTORES NORMAL MICROVASCULAR PERMEABILITY IN Cav-1 NULL MICE

Microvascular permeability is mediated by (i) the caveolar transcytosis of molecules across endothelial cells and (ii) the paracellular movement of ions and nutrients. Recently, we derived Cav-1 (−/−) knock-out mice using standard homologous recombination techniques. These mice are viable but show a loss of endothelial cell caveolae and striking defects in caveolae-mediated endocytosis. Thus, a compensatory mechanism must be operating in these mice. One possible compensatory response would be an increase in the paracellular pathway, resulting in increased microvascular permeability. To test this hypothesis directly, we studied the microvascular permeability of Cav-1 null mice using a variety of complementary in vivo approaches. Radio-iodinated bovine serum albumin was injected into Cav-1-deficient mice, and its rate of clearance from the circulatory system was compared with that of wild type control mice. Our results indicate that iodinated bovine serum albumin is removed from the circulatory system of Cav-1-deficient mice at a substantially faster rate. To determine whether this defect is restricted to the paracellular movement of albumin, lungs from Cav-1-deficient mice were next perfused with the electron dense dye Ruthenium Red. Micrographs of lung endothelial cells from Cav-1-deficient mice demonstrate that the paracellular movement of Ruthenium Red is dramatically increased. In addition, electron micrographs of Cav-1-deficient lung capillaries reveal defects in tight junction morphology and abnormalities in capillary endothelial cell adhesion to the basement membrane. This defect in cell-substrate attachment is consistent with the postulated role of caveolin-1 in positively regulating integrin signaling. Because loss of caveolin-1 expression results in constitutive activation of eNOS activity, we also examined whether these increases in microvascular permeability are NO-dependent. Interestingly, treatment with l-NAME (a well established nitric-oxide synthase inhibitor) successfully reversed the microvascular hyperpermeability phenotype of Cav-1 knock-out mice. Thus, caveolin-1 plays a dual regulatory role in controlling microvascular permeability: (i) as a structural protein that is required for caveolae formation and caveolar transcytosis and (ii) as a tonic inhibitor of eNOS activity to negatively regulate the paracellular pathway.

Caveolin-1/3 Double-Knockout Mice Are Viable, but Lack Both Muscle and Non-Muscle Caveolae, and Develop a Severe Cardiomyopathic Phenotype

The caveolin gene family consists of caveolins 1, 2, and 3. Caveolins 1 and 2 are co-expressed in many cell types, such as endothelial cells, fibroblasts, smooth muscle cells and adipocytes, where they form a heteroligomeric complex. In contrast, the expression of caveolin-3 is muscle-specific. Thus, the expression of caveolin-1 is required for caveolae formation in non-muscle cells, while the expression of caveolin-3 drives caveolae formation in striated muscle cell types (cardiac and skeletal). To create a truly caveolae-deficient mouse, we interbred Cav-1 null mice and Cav-3 null mice to generate Cav-1/Cav-3 double-knockout (Cav-1/3 dKO) mice. Here, we report that Cav-1/3 dKO mice are viable and fertile, despite the fact that they lack morphologically identifiable caveolae in endothelia, adipocytes, smooth muscle cells, skeletal muscle fibers, and cardiac myocytes. We also show that these mice are deficient in all three caveolin gene products, as caveolin-2 is unstable in the absence of caveolin-1. Interestingly, Cav-1/3 dKO mice develop a severe cardiomyopathy. At 2 months of age, analysis of Cav-1/3 dKO hearts via gated magnetic resonance imaging reveals a dramatic increase in left ventricular wall thickness, as compared with Cav-1-KO, Cav-3 KO, and wild-type mice. Further functional analysis of Cav-1/3 dKO hearts via transthoracic echocardiography demonstrates hypertrophy and dilation of the left ventricle, with a significant decrease in fractional shortening. As predicted, Northern analysis of RNA derived from the left ventricle of Cav-1/3 dKO mice shows a dramatic up-regulation of the atrial natriuretic factor message, a well-established biochemical marker of cardiac hypertrophy. Finally, histological analysis of Cav-1/3 dKO hearts reveals hypertrophy, disorganization, and degeneration of the cardiac myocytes, as well as chronic interstitial fibrosis and inflammation. Thus, dual ablation of both Cav-1 and Cav-3 genes in mice leads to a pleiotropic defect in caveolae formation and severe cardiomyopathy.

Absence of Caveolin-1 Sensitizes Mouse Skin to Carcinogen-Induced Epidermal Hyperplasia and Tumor Formation

Caveolin-1 is the principal protein component of caveolae membrane domains, which are located at the cell surface in most cell types. Evidence has accumulated suggesting that caveolin-1 may function as a suppressor of cell transformation in cultured cells. The human CAV-1 gene is located at a putative tumor suppressor locus (7q31.1/D7S522) and a known fragile site (FRA7G) that is deleted in a variety of epithelial-derived tumors. Mechanistically, caveolin-1 is known to function as a negative regulator of the Ras-p42/44 MAP kinase cascade and as a transcriptional repressor of cyclin D1, possibly explaining its transformation suppressor activity in cultured cells. However, it remains unknown whether caveolin-1 functions as a tumor suppressor gene in vivo. Here, we examine the tumor suppressor function of caveolin-1 using Cav-1 (-/-) null mice as a model system. Cav-1 null mice and their wild-type counterparts were subjected to carcinogen-induced skin tumorigenesis, using 7,12-dimethylbenzanthracene (DMBA). Mice were monitored weekly for the development of tumors. We demonstrate that Cav-1 null mice are dramatically more susceptible to carcinogen-induced tumorigenesis, as they develop skin tumors at an increased rate. After 16 weeks of DMBA-treatment, Cav-1 null mice showed a 10-fold increase in tumor incidence, a 15-fold increase in tumor number per mouse (multiplicity), and a 35-fold increase in tumor area per mouse, as compared with wild-type littermate mice. Moreover, before the development of tumors, DMBA-treatment induced severe epidermal hyperplasia in Cav-1 null mice. Both the basal cell layer and the suprabasal cell layers were expanded in treated Cav-1 null mice, as evidenced by immunostaining with cell-type specific differentiation markers (keratin-10 and keratin-14). In addition, cyclin D1 and phospho-ERK1/2 levels were up-regulated during epidermal hyperplasia, suggesting a possible mechanism for the increased susceptibility of Cav-1 null mice to tumorigenesis. However, the skin of untreated Cav-1 null mice appeared normal, without any evidence of epidermal hyperplasia, despite the fact that Cav-1 null keratinocytes failed to express caveolin-1 and showed a complete ablation of caveolae formation. Thus, Cav-1 null mice require an appropriate oncogenic stimulus, such as DMBA treatment, to reveal their increased susceptibility toward epidermal hyperplasia and skin tumor formation. Our results provide the first genetic evidence that caveolin-1 indeed functions as a tumor suppressor gene in vivo.

The adipocyte as an important target cell for Trypanosoma cruzi infection

Adipose tissue plays an active role in normal metabolic homeostasis as well as in the development of human disease. Beyond its obvious role as a depot for triglycerides, adipose tissue controls energy expenditure through secretion of several factors. Little attention has been given to the role of adipocytes in the pathogenesis of Chagas disease and the associated metabolic alterations. Our previous studies have indicated that hyperglycemia significantly increases parasitemia and mortality in mice infected with Trypanosoma cruzi. We determined the consequences of adipocyte infection in vitro and in vivo. Cultured 3T3-L1 adipocytes can be infected with high efficiency. Electron micrographs of infected cells revealed a large number of intracellular parasites that cluster around lipid droplets. Furthermore, infected adipocytes exhibited changes in expression levels of a number of different adipocyte-specific or adipocyte-enriched proteins. The adipocyte is therefore an important target cell during acute Chagas disease. Infection of adipocytes by T. cruzi profoundly influences the pattern of adipokines. During chronic infection, adipocytes may represent an important long-term reservoir for parasites from which relapse of infection can occur. We have demonstrated that acute infection has a unique metabolic profile with a high degree of local inflammation in adipose tissue, hypoadiponectinemia, hypoglycemia, and hypoinsulinemia but with relatively normal glucose disposal during an oral glucose tolerance test.

Caveolin-1 Knockout Mice Show an Impaired Angiogenic Response to Exogenous Stimuli

Recent studies have shown that caveolin-1 (Cav-1) plays an important role as a regulator of angiogenesis in vitro. Here, we use Cav-1 knockout (KO) mice as a model system to examine the in vivo relevance of these findings. A primary mediator of angiogenesis is basic fibroblast growth factor (bFGF). Thus, we studied bFGF-induced angiogenesis in Cav-1 KO mice using a reconstituted basement membrane system, ie, Matrigel plugs, supplemented with bFGF. In Cav-1 KO mice, implanted Matrigel plugs showed a dramatic reduction in both vessel infiltration and density, as compared with identical plugs implanted in wild-type control mice. We also examined the necessity of Cav-1 to support the angiogenic response of an exogenous tumor by subcutaneously injecting Cav-1 KO mice with the melanoma cell line, B16-F10. We show that tumor weight, volume, and vessel density are all reduced in Cav-1 KO mice, consistent with diminished angiogenesis. Ultrastructural analysis of newly formed capillaries within the exogenous tumors reveals a lack of endothelial caveolae and incomplete capillary formation in Cav-1 KO mice. These results provide novel evidence that Cav-1 and caveolae play an important positive role in the process of pathological angiogenesis in vivo.

The M Cell as a Portal of Entry to the Lung for the Bacterial Pathogen Mycobacterium tuberculosis

M. tuberculosis accesses the terminal lung and is phagocytosed by alveolar macrophages. Utilizing a mouse intratracheal challenge model, we demonstrate that M. tuberculosis rapidly enters through M cells as well. From there, bacilli are deposited within associated intraepithelial leukocytes and subsequently conveyed to the draining lymph nodes early after infection. Osteopetrotic (Csfm(op)/Csfm(op)) mice, null mutants for macrophage colony-stimulating factor, possess diminished numbers of circulating monocytes and tissue macrophages. Csfm(op)/Csfm(op) mice were highly susceptible to challenge with M. tuberculosis. In contrast to controls, tubercle bacilli were not conveyed to draining lymph nodes early after infection but were instead retained within the mucosa. These results indicate that M cells represent an alternate portal of entry for M. tuberculosis, which may contribute to the rapid development of protective lung immune responses.

Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is absent in the skeletal muscle of DMD patients and mdx mice. At the plasma membrane of skeletal muscle fibers, dystrophin associates with a multimeric protein complex, termed the dystrophin-glycoprotein complex (DGC). Protein members of this complex are normally absent or greatly reduced in dystrophin-deficient skeletal muscle fibers, and are thought to undergo degradation through an unknown pathway. As such, we reasoned that inhibition of the proteasomal degradation pathway might rescue the expression and subcellular localization of dystrophin-associated proteins. To test this hypothesis, we treated mdx mice with the well-characterized proteasomal inhibitor MG-132. First, we locally injected MG-132 into the gastrocnemius muscle, and observed the outcome after 24 hours. Next, we performed systemic treatment using an osmotic pump that allowed us to deliver different concentrations of the proteasomal inhibitor, over an 8-day period. By immunofluorescence and Western blot analysis, we show that administration of the proteasomal inhibitor MG-132 effectively rescues the expression levels and plasma membrane localization of dystrophin, beta-dystroglycan, alpha-dystroglycan, and alpha-sarcoglycan in skeletal muscle fibers from mdx mice. Furthermore, we show that systemic treatment with the proteasomal inhibitor 1) reduces muscle membrane damage, as revealed by vital staining (with Evans blue dye) of the diaphragm and gastrocnemius muscle isolated from treated mdx mice, and 2) ameliorates the histopathological signs of muscular dystrophy, as judged by hematoxylin and eosin staining of muscle biopsies taken from treated mdx mice. Thus, the current study opens new and important avenues in our understanding of the pathogenesis of DMD. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD.

Molecular Mapping and Functional Characterization of the VEGF164 Heparin-binding Domain

The longer splice isoforms of vascular endothelial growth factor-A (VEGF-A), including mouse VEGF164, contain a highly basic heparin-binding domain (HBD), which imparts the ability of these isoforms to be deposited in the heparan sulfate-rich extracellular matrix and to interact with the prototype sulfated glycosaminoglycan, heparin. The shortest isoform, VEGF120, lacks this highly basic domain and is freely diffusible upon secretion. Although the HBD has been attributed significant relevance to VEGF-A biology, the molecular determinants of the heparin-binding site are unknown. We used site-directed mutagenesis to identify amino acid residues that are critical for heparin binding activity of the VEGF164 HBD. We focused on basic residues and found Arg-13, Arg-14, and Arg-49 to be critical for heparin binding and interaction with extracellular matrix in tissue samples. We also examined the cellular and biochemical consequences of abolishing heparin-binding function, measuring the ability of the mutants to interact with VEGF receptors, induce endothelial cell gene expression, and trigger microvessel outgrowth. Induction of tissue factor expression, vessel outgrowth, and binding to VEGFR2 were unaffected by the HBD mutations. In contrast, the HBD mutants showed slightly decreased binding to the NRP1 (neuropilin-1) receptor, and analyses suggested the heparin and NRP1 binding sites to be distinct but overlapping. Finally, mutations that affect the heparin binding activity also led to an unexpected reduction in the affinity of VEGF164 binding specifically to VEGFR1. This finding provides a potential basis for previous observations suggesting enhanced potency of VEGF164 versus VEGF120 in VEGFR1-mediated signaling in inflammatory cells.

Requirement of transcription factor NFAT in developing atrial myocardium.

Nuclear factor of activated T cell (NFAT) is a ubiquitous regulator involved in multiple biological processes. Here, we demonstrate that NFAT is temporally required in the developing atrial myocardium between embryonic day 14 and P0 (birth). Inhibition of NFAT activity by conditional expression of dominant-negative NFAT causes thinning of the atrial myocardium. The thin myocardium exhibits severe sarcomere disorganization and reduced expression of cardiac troponin-I (cTnI) and cardiac troponin-T (cTnT). Promoter analysis indicates that NFAT binds to and regulates transcription of the cTnI and the cTnT genes. Thus, regulation of cytoskeletal protein gene expression by NFAT may be important for the structural architecture of the developing atrial myocardium.

Identification and characterization of a cell surface marker for embryonic rat spinal accessory motor neurons.

The developing mammalian spinal cord contains distinct populations of motor neurons that can be distinguished by their cell body positions, by the expression of specific combinations of regulatory genes, and by the paths that their axons take to exit the central nervous system (CNS). Subclasses of spinal motor neurons are also thought to express specific cell surface proteins that function as receptors which control the guidance of their axons. We identified monoclonal antibody (mAb) SAC1 in a screen aimed at generating markers for specific subsets of neurons/axons in the developing rat spinal cord. During early embryogenesis, mAb SAC1 selectively labels a small subset of Isl1‐positive motor neurons located exclusively within cervical segments of the spinal cord. Strikingly, these neurons extend mAb SAC1‐positive axons along a dorsally directed trajectory toward the lateral exit points. Consistent with the finding that mAb SAC1 also labels spinal accessory nerves, these observations identify mAb SAC1 as a specific marker of spinal accessory motor neurons/axons. During later stages of embryogenesis, mAb SAC1 is transiently expressed on both dorsally and ventrally projecting spinal motor neurons/axons. Interestingly, mAb SAC1 also labels the notochord and floor plate during most stages of spinal cord development. The mAb SAC1 antigen is a 100‐kD glycoprotein that is likely to be the rat homolog of SC1/BEN/DM‐GRASP, a homophilic adhesion molecule that mediates axon outgrowth and fasciculation. J. Comp. Neurol. 439:368–383, 2001.