Andrew Tri Van Ho

Cellular turnover and extracellular matrix remodeling in female reproductive tissues: functions of metalloproteinases and their inhibitors.

Abstract. Female reproductive tissues possess a unique ability to accommodate a remarkable amount of cell turnover and extracellular matrix (ECM) remodeling following puberty. Cellular structures within ovary, uterus, and mammary tissue not only change cyclically in response to ovarian hormones but also undergo differentiation during pregnancy, and eventually revert to that resembling the pre-pregnant stage. Cell proliferation, apoptosis, invasion, and differentiation are integral cellular processes that are precisely regulated in reproductive tissues, but become dysregulated in pathologies such as cancer. Explicit reorganization of ECM and basement membranes is also critical to preserve the form and function of these tissues. Here we review the evidence that coordinated spatiotemporal expression patterns of matrix metalloproteinase (MMP) genes and their tissue inhibitors (TIMPs) are important in cell and ECM turnover of the ovary, uterus, and mammary tissues. We discuss how perturbation in these gene families may impact the biology of these reproductive tissues and the factors implicated in the control of MMP and TIMP gene expression. The observed trends in MMP and TIMP expression involved in ovarian and mammary carcinomas are also presented.

Host TIMP-1 overexpression confers resistance to experimental brain metastasis of a fibrosarcoma cell line

Within the tumor-stromal microenvironment a disrupted balance between matrix metalloproteinases (MMPs) and their inhibitors compromises the integrity of the extracellular matrix and promotes malignancy. Tissue inhibitors of metalloproteinases (TIMPs) have been linked to tumor suppression in studies of genetically altered tissue culture cells and in analyses of clinical specimens in situ. We generated transgenic mice as a model system to test the relationship between TIMP-1 levels in a host organ and susceptibility to experimentally targeted metastasis. Ectopically overexpressed TIMP-1 in the brain resulted in a tissue microenvironment with elevated protein levels of this natural MMP inhibitor. Metastatic challenge provided by lacZ-tagged fibrosarcoma cells permitted high-resolution analysis of metastatic load and pattern. We found that elevated host TIMP-1 imposed resistance to experimental metastasis of fibrosarcoma: In TIMP-1 overexpressing mice, brain metastases were significantly reduced by 75% compared to wild-type littermates. Our findings demonstrate that ectopic TIMP-1 expression efficiently exerts a suppressive effect on metastasizing tumor cells.

The central role of muscle stem cells in regenerative failure with aging

Skeletal muscle mass, function, and repair capacity all progressively decline with aging, restricting mobility, voluntary function, and quality of life. Skeletal muscle repair is facilitated by a population of dedicated muscle stem cells (MuSCs), also known as satellite cells, that reside in anatomically defined niches within muscle tissues. In adult tissues, MuSCs are retained in a quiescent state until they are primed to regenerate damaged muscle through cycles of self-renewal divisions. With aging, muscle tissue homeostasis is progressively disrupted and the ability of MuSCs to repair injured muscle markedly declines. Until recently, this decline has been largely attributed to extrinsic age-related alterations in the microenvironment to which MuSCs are exposed. However, as highlighted in this Perspective, recent reports show that MuSCs also progressively undergo cell-intrinsic alterations that profoundly affect stem cell regenerative function with aging. A more comprehensive understanding of the interplay of stem cell–intrinsic and extrinsic factors will set the stage for improving cell therapies capable of restoring tissue homeostasis and enhancing muscle repair in the aged.

Pathologic calcification of adult vascular smooth muscle cells differs on their crest or mesodermal embryonic origin

Vascular calcifications can occur in the elderly and in patients suffering from various diseases. Interestingly, depending on the pathology, different regions of the arterial system can be affected. Embryonic observations have clearly indicated that vascular smooth muscle cell (VSMC) origin is notably heterogeneous. For instance, in the aorta, VSMCs colonizing the aortic arch region derive from cardiac neural crest cells, whereas those populating the descending aorta derive from the mesoderm. We examined here whether the embryonic origin of aortic VSMCs would correlate with their ability to mineralize. Under hyperphosphatemic conditions that induce vascular calcifications, we performed ex vivo aortic explant cultures as well as in vitro VSMC cultures from wild‐type mice. Our data showed that VSMC embryonic origin affects their ability to mineralize. Indeed, the aortic arch media made up of VSMCs of neural crest origin calcifies significantly earlier than the descending aorta composed of VSMCs, which are mesoderm‐derived. Similar results were obtained with cultured VSMCs harvested from both aortic regions. We also demonstrated that in a mouse model deficient in matrix Gla protein, a potent calcification inhibitor, developing extensive and spontaneous medial calcifications of the aorta, lesions initiate in the aortic arch. Subsequently, calcifications progress outside the aortic arch region and ultimately spread all over the entire arterial tree, including the descending aorta. Altogether, our results support an unsuspected correlation between VSMCs of embryonic origin and the timing of appearance of calcifications.

Coupling of caspase‐9 to Apaf1 in response to loss of pRb or cytotoxic drugs is cell‐type‐specific

Inactivation of the tumor suppressor Rb in the mouse induces cell death, which depends entirely (in lens, CNS) and only partly (PNS, skeletal muscles) on Apaf1/Ced4, an apoptosomal factor thought to be required for processing procaspase‐9 following mitochondrial permeabilization. Here, we report that in response to cytotoxic drugs, Apaf1−/− primary myoblasts but not fibroblasts undergo bona fide apoptosis. Cell demise was associated with disruption of mitochondria but not endoplasmic reticulum. Processing of procaspase‐9 occurred in Apaf1−/− myoblasts but not fibroblasts, and ablation of Casp9 prevented drug‐induced apoptosis in both cell types. Deregulation of the Rb pathway by overexpression of E2F1 also induced caspase‐9‐dependent, Apaf1‐independent apoptosis in myoblasts. Despite its requirement for apoptosis in vitro, mutation in Casp9 abrogated cell death in the nervous system and lens but only partly in skeletal muscles of Rb‐deficient embryos. In addition, developmental cell death in fetal liver and PNS was not inhibited in Casp9−/− embryos. Therefore, loss of pRb elicits apoptosome‐dependent and apoptosome‐independent cell death, and the requirement and coupling of caspase‐9 to Apaf1 are both context‐dependent.

Neural Crest Cell Lineage Restricts Skeletal Muscle Progenitor Cell Differentiation through Neuregulin1-ErbB3 Signaling

Coordinating the balance between progenitor self-renewal and myogenic differentiation is required for a regulated expansion of the developing muscles. Previous observation that neural crest cells (NCCs) migrate throughout the somite regions, where trunk skeletal muscles first emerge, suggests a potential role for these cells in influencing early muscle formation. However, specific signaling interactions between NCCs and skeletal muscle cells remain unknown. Here we show that mice with specific NCC and peripheral nervous system defects display impaired survival of skeletal muscle and show skeletal muscle progenitor cell (MPC) depletion due to precocious commitment to differentiation. We show that reduced NCC-derived Neuregulin1 (Nrg1) in the somite region perturbs ErbB3 signaling in uncommitted MPCs. Using a combination of explant culture experiments and genetic ablation in the mouse, we demonstrate that Nrg1 signals provided by the NCC lineage play a critical role in sustainable myogenesis, by restraining MPCs from precocious differentiation.

Proteolysis during tumor cell extravasation in vitro: metalloproteinase involvement across tumor cell types.

To test if proteolysis is involved in tumor cell extravasation, we developed an in vitro model where tumor cells cross an endothelial monolayer cultured on a basement membrane. Using this model we classified the ability of the cells to transmigrate through the endothelial cell barrier onto the underlying matrix, and scored this invasion according to the stage of passage through the endothelium. Metalloproteinase inhibitors reduced tumor cell extravasation by at least 35%. Visualization of protease and cell adhesion molecules by confocal microscopy demonstrated the cell surface localization of MMP-2, MMP-9, MT1-MMP, furin, CD44 and αvβ3, during the process of transendothelial migration. By the addition of inhibitors and bio-modulators we assessed the functional requirement of the aforementioned molecules for efficient migration. Proteolytic digestion occurred at the cell-matrix interface and was most evident during the migratory stage. All of the inhibitors and biomodulators affected the transition of the tumor cells into the migratory stage, highlighting the most prevalent use of proteolysis at this particular step of tumor cell extravasation. These data suggest that a proteolytic interface operates at the tumor cell surface within the tumor-endothelial cell microenvironment.

Splitting the apoptosome.

Assembly of the apoptosome in response to mitochondrial permeabilization, the hallmark of the intrinsic apoptotic pathway, involves binding of cytochrome c to Apaf1, recruitment and auto-processing of the apical/signaling pro-caspase-9, and coupled activation of downstream/executioner caspases like caspase 3. Evidence now indicates that certain apoptotic cascades can bypass the apoptosome and activate caspase-9 independent of the mitochondria. Recently, we have demonstrated that caspase-9 can be activated in Apaf1-mutant primary myoblasts, but not fibroblasts, in response to stimuli that are known to act via the mitochondria. Thus, apoptosomal activation of caspase-9 seems to represent only one of the routes for its activation; other pathways, some of which are yet to be discovered, can bypass the requirement for Apaf1 and activate caspase-9 in a tissue and context specific manner.

XIAP Activity Dictates Apaf-1 Dependency for Caspase 9 Activation

ABSTRACT The current model for the intrinsic apoptotic pathway holds that mitochondrial activation of caspases in response to cytotoxic drugs requires both Apaf-1-induced dimerization of procaspase 9 and Smac/Diablo-mediated sequestration of inhibitors of apoptosis proteins (IAPs). Here, we showed that either pathway can independently promote caspase 9 activation in response to apoptotic stimuli. In drug-treated Apaf-1−/− primary myoblasts, but not fibroblasts, Smac/Diablo accumulates in the cytosol and sequesters X-linked IAP (XIAP), which is expressed at lower levels in myoblasts than in fibroblasts. Consequently, caspase 9 activation proceeds in Apaf-1−/− myoblasts; concomitant ablation of Apaf-1 and Smac is required to prevent caspase 9 activation and the onset of apoptosis. Conversely, in stimulated Apaf-1−/− fibroblasts, the ratio of XIAP to Smac/Diablo is high compared to that for myoblasts and procaspase 9 is not activated. Suppressing XIAP with exogenous Smac/Diablo or a pharmacological inhibitor can still induce caspase 9 in drug-treated Apaf-1-null fibroblasts. Thus, caspase 9 activation in response to intrinsic apoptotic stimuli can be uncoupled from Apaf-1 in vivo by XIAP antagonists.

Critical role of the Rb family in myoblast survival and fusion.

The tumor suppressor Rb is thought to control cell proliferation, survival and differentiation. We recently showed that differentiating Rb-deficient mouse myoblasts can fuse to form short myotubes that quickly collapse through a mechanism involving autophagy, and that autophagy inhibitors or hypoxia could rescue the defect leading to long, twitching myotubes. Here we determined the contribution of pRb relatives, p107 and p130, to this process. We show that chronic or acute inactivation of Rb plus p107 or p130 increased myoblast cell death and reduced myotube formation relative to Rb loss alone. Treatment with autophagy antagonists or hypoxia extended survival of double-knockout myotubes, which appeared indistinguishable from control fibers. In contrast, triple mutations in Rb, p107 and p130, led to substantial increase in myoblast death and to elongated bi-nuclear myocytes, which seem to derive from nuclear duplication, as opposed to cell fusion. Under hypoxia, some rare, abnormally thin triple knockout myotubes survived and twitched. Thus, mutation of p107 or p130 reduces survival of Rb-deficient myoblasts during differentiation but does not preclude myoblast fusion or necessitate myotube degeneration, whereas combined inactivation of the entire Rb family produces a distinct phenotype, with drastically impaired myoblast fusion and survival.