A proteomics analysis of the effects of chronic hemiparetic stroke on troponin T expression in human vastus lateralis.

Stroke disability is attributed to upper motor neuron deficits resulting from ischemic brain injury. We have developed proteome maps of the Vastus lateralis to examine the effects of ischemic brain injury on paretic skeletal muscle myofilament proteins. Proteomics analyses from seven hemiparetic stroke patients have detected a decrease of three troponin T isoforms in the paretic muscle suggesting that myosin-actin interactions may be attenuated. We propose that ischemic brain injury may prevent troponin T participation in complex formation thereby affecting the protein interactions associated with excitation-contraction coupling. We have also detected a novel skeletal troponin T isoform that has a C-terminal variation. Our data suggest that the decreased slow troponin T isoform pools in the paretic limb may contribute to the gait deficit after stroke. The complexity of the neurological deficit on Vastus lateralis is suggested by the multiple changes in proteins detected by our proteomics mapping.

Cell surface proteomics identifies molecules functionally linked to tumor cell intravasation.

In order to better understand the molecular and cellular determinants of tumor cell intravasation, our laboratory has generated a pair of congenic human HT-1080 fibrosarcoma variants (i.e. HT-hi/diss and HT-lo/diss) differing 50-100-fold in their ability to intravasate and disseminate. To investigate the molecular differences underlying the distinct dissemination capacities of these HT-1080 variants, we performed a comparative analysis of the cell surface proteomes of HT-hi/diss and HT-lo/diss. Cell membrane proteins were enriched by biotinylation and avidin precipitation and analyzed by tandem mass spectrometry employing multidimensional protein identification technology. By this approach, 47 cell surface-associated molecules were identified as differentially expressed between the HT-1080 intravasation variants. From these candidates, four targets (i.e. TIMP-2, NCAM-1, JAM-C, and tissue factor (TF)) were selected for further biochemical validation and in vivo functional verification. Western blot analysis of the cell surface enriched fractions confirmed the proteomic array data, demonstrating that, in vitro, TIMP-2 protein was increased in the HT-lo/diss variant, whereas NCAM-1, JAM-C, and TF levels were increased in the HT-hi/diss variant. Corresponding in vivo differences in levels of TIMP-2, JAM-C, and TF were demonstrated in primary tumors grown in the chick embryo. Finally, functional inhibition of one selected protein (i.e. TF) by small interfering RNA silencing or ligation with a function-blocking antibody significantly reduced HT-hi/diss intravasation, thus clearly implicating TF in the early steps of tumor cell dissemination. Overall, our cell surface proteomic analysis provides a powerful tool for identification of specific cell membrane molecules that contribute functionally to intravasation and metastasis in vivo.

Functional analysis of matrix metalloproteinases and tissue inhibitors of metalloproteinases differentially expressed by variants of human HT-1080 fibrosarcoma exhibiting high and low levels of intravasation and metastasis.

The role of tumor-derived matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) in cancer cell dissemination was analyzed by employing two variants of human HT-1080 fibrosarcoma, HT-hi/diss and HT-lo/diss, which differ by 50-100-fold in their ability to intravasate and metastasize in the chick embryo. HT-hi/diss and HT-lo/diss were compared by quantitative reverse transcription-PCR and Western blot analyses for mRNA and protein expression of nine MMPs (MMP-1, -2, -3, -7, -8, -9, -10, -13, and -14) and three TIMPs (TIMP-1, -2, and -3) in cultured cells in vitro and in primary tumors in vivo. MMP-1 and MMP-9 were more abundant in the HT-hi/diss variant, both in cultures and in tumors, whereas the HT-lo/diss variant consistently expressed higher levels of MMP-2, TIMP-1, and TIMP-2. Small interfering RNA-mediated down-regulation of MMP-2 and TIMP-2 increased intravasation of HT-lo/diss cells. Coordinately, treatment of the developing HT-hi/diss tumors with recombinant TIMP-1 and TIMP-2 significantly reduced HT-hi/diss cell intravasation. However, a substantial increase of HT-hi/diss dissemination was observed upon small interfering RNA-mediated down-regulation of three secreted MMPs, including the interstitial collagenase MMP-1 and the two gelatinases, MMP-2 and MMP-9, but not the membrane-tethered MMP-14. The addition of recombinant pro-MMP-9 protein to the HT-hi/diss tumors reversed the increased intravasation of HT-hi/diss cells, in which MMP-9 was stably down-regulated by short hairpin RNA interference. This rescue did not occur if the pro-MMP-9 was stoichiometrically complexed with TIMP-1, pointing to a direct role of the MMP-9 enzyme in regulation of HT-hi/diss intravasation. Collectively, these findings demonstrate that tumor-derived MMPs may have protective functions in cancer cell intravasation, i.e. not promoting but rather catalytically interfering with the early stages of cancer dissemination.

Activity-based protein profiling implicates urokinase activation as a key step in human fibrosarcoma intravasation.

Entry of malignant cells into the vasculature (i.e. intravasation) requires proteolytic remodeling of the extracellular matrix so that tumor cells may pass through the local stroma and penetrate the vessel wall. The circulatory system then provides a means of transporting tumor cells to distant sites where they extravasate and establish metastatic lesions. This study utilizes activity-based protein profiling to compare the active serine hydrolase repertoire in high intravasating (HT-hi/diss) and low intravasating (HT-lo/diss) variants of the human fibrosarcoma HT-1080 cell line to determine which enzyme(s) play a role in intravasation. Activity-based protein profiling revealed multiple serine hydrolases with altered activity between HT-hi/diss and HT-lo/diss cells, with the largest difference being the activity of urokinase-type plasminogen activator (uPA). Levels of inactive uPA zymogen were similar between the two cell variants, but only HT-hi/diss conditioned medium contained active uPA, suggesting that uPA activation may contribute to the enhanced intravasation of HT-hi/diss cells. To analyze the role of uPA activity specifically in the process of intravasation, we grafted cells from the two HT-1080 variants onto the chorioallantoic membrane of chick embryos and measured levels of tumor cell intravasation in the distal chorioallantoic membrane using quantitative human-specific Alu PCR. Inhibition of uPA activity with natural (plasminogen activator inhibitor-1) or synthetic (amiloride) inhibitors diminished HT-hi/diss Matrigel invasion in vitro and intravasation and metastasis in vivo. Additionally, treatment of HT-lo/diss tumors with exogenous active uPA increased the number of intravasated cells in vivo. These results indicate that active uPA promotes tumor cell intravasation and that uPA activation appears to be a key step in tumor progression.

Proangiogenic role of neutrophil-like inflammatory heterophils during neovascularization induced by growth factors and human tumor cells.

A quantitative in vivo angiogenesis model employing collagen onplants placed on the chick embryo chorioallantoic membrane (CAM) has been used in this study to assess the spatial and temporal associations between neutrophil-like inflammatory cells, namely chicken heterophils, and the development of new blood vessels. Previously we have demonstrated that monocytes/macrophages infiltrating the onplants were associated with extracellular matrix remodeling and angiogenesis, in particular by delivering MMP-13 collagenase. By introducing chicken gelatinase B (chMMP-9) as a specific marker for heterophils, we now show that the onset and extent of angiogenesis induced by purified growth factors or by human HT-1080 fibrosarcoma cells correlated with the initial influx of chMMP-9-positive heterophils. This early heterophil arrival was followed by the infiltration of monocytes/macrophages and appeared to sustain further blood vessel formation. The disruption of inflammatory cell influx by 2 mechanistically distinct anti-inflammatory drugs, cortisone and ibuprofen, significantly inhibited angiogenesis, indicating a functional involvement of these inflammatory cells in new blood vessel development. A direct addition of isolated heterophils or purified chMMP-9 into the HT-1080 onplants engrafted into cortisone- or ibuprofen-treated embryos reversed the antiangiogenic effects of the drugs. The exogenously added heterophils induced in vivo a further infiltration of endogenous heterophils and monocytes and dramatically rescued the impaired angiogenesis, highlighting the importance of early inflammatory leukocytes in tumor-induced angiogenesis. Moreover, purified heterophils incorporated into onplants lacking growth factors or tumor cells induced angiogenesis in nontreated embryos, further indicating a direct proangiogenic role for neutrophil-like leukocytes.

Unexpected effect of matrix metalloproteinase down-regulation on vascular intravasation and metastasis of human fibrosarcoma cells selected in vivo for high rates of dissemination.

The human tumor/chick embryo model involving grafting of human HT-1080 fibrosarcoma cells on the chorioallantoic membrane was used in conjunction with quantitative real-time Alu PCR to select in vivo a pair of isogenic cell lines (HT-hi/diss and HT-lo/diss), dramatically differing in their ability to disseminate from the primary tumor (i.e., intravasate into the chorioallantoic membrane vasculature and metastasize to the lungs). During an immunohistochemical time course study, HT-hi/diss cells were sequentially visualized having escaped from the primary tumors, engaged with the blood vessels, and eventually observed inside the chorioallantoic membrane capillaries, thus reflecting early intravasating events. In contrast, HT-lo/diss cells seemed restricted to their primary tumor. Importantly, after i.v. inoculation, both variants arrested, extravasated, and proliferated in host tissues with similar efficiencies, highlighting that the observed earlier events at the periphery of the primary tumor could account for their differential dissemination. In a mechanistic probing of these events, we determined that HT-hi/diss intravasation was sensitive to a broad-range matrix metalloproteinase (MMP) inhibitor. To analyze the possible role of individual MMPs, membrane-bound MMP-14 and secreted MMP-9 were individually down-regulated in HT-hi/diss cells with their corresponding small interfering RNAs. Despite efficient down-regulation of MMP-14, neither intravasation nor metastasis of HT-hi/diss cells was affected significantly. However, a substantial down-regulation of MMP-9 was accompanied by a surprising 3-fold increase in intravasation and metastasis. The results emphasize a rising awareness that targeting certain MMPs might result in an enhanced malignancy, exemplified herein at the intravasation level as this step of the metastatic cascade is dissected and quantified.

Altered oxidative stress response of the long-lived Snell dwarf mouse.

Several single gene mutations in mice that increase the murine life span have been identified, including the Pit-1 mutation which results in the Snell dwarf (Pit1(dw/dw)), however, the biological mechanism of this life-span extension is still unclear. Based on studies that show oxidative stress plays an important role in the aging process, we hypothesized that the increased longevity seen in Snell dwarf mice may result from a resistance to oxidative stress. We report that Snell dwarf mice respond to oxidative stress induced by 3-NPA differently than their wild type littermates. This altered response results in diminished activation of the MEK-ERK kinase cascade and virtually no phosphorylation of c-Jun at Ser63 in dwarf mice after 3-NPA treatment, despite a robust phosphorylation of Ser63 in wild type mice. We propose that this altered management of oxidative stress in dwarf mice is partially responsible for the increased longevity in Snell dwarf mice.