Proteomic Profiling of Mesenchymal Stem Cell Responses to Mechanical Strain and TGF-β1

Mesenchymal stem cells (MSCs) are a potential source of smooth muscle cells (SMCs) for constructing tissue-engineered vascular grafts. However, the details of how specific combinations of vascular microenvironmental factors regulate MSCs are not well understood. Previous studies have suggested that both mechanical stimulation with uniaxial cyclic strain and chemical stimulation with transforming growth factor-β1 (TGF-β1) can induce smooth muscle markers in MSCs. In this study, we investigated the combined effects of uniaxial cyclic strain and TGF-β1 stimulation on MSCs. By using a proteomic analysis, we found differential regulation of several proteins and genes, such as the up-regulation of TGF-β1-induced protein ig-h3 (BGH3) protein levels by TGF-β1 and up-regulation of calponin 3 protein level by cyclic strain. At the gene expression level, BGH3 was induced by TGF-β1, but calponin 3 was not significantly regulated by mechanical strain or TGF-β1, which was in contrast to the synergistic up-regulation of calponin 1 gene expression by cyclic strain and TGF-β1. Further experiments with cycloheximide treatment suggested that the up-regulation of calponin 3 by cyclic strain was at post-transcriptional level. The results in this study suggest that both mechanical stimulation and TGF-β1 signaling play unique and important roles in the regulation of MSCs at both transcriptional and post-transcriptional levels, and that a precise combination of microenvironmental cues may promote MSC differentiation

Dosimetric evaluation and radioimmunotherapy of anti-tumour multivalent Fab́ fragments

We have been investigating the use of cross-linked divalent (DFM) and trivalent (TFM) versions of the anti-carcinoembryonic antigen (CEA) monoclonal antibody A5B7 as possible alternatives to the parent forms (IgG and F(ab́)2) which have been used previously in clinical radioimmunotherapy (RIT) studies in colorectal carcinoma. Comparative biodistribution studies of similar sized DFM and F(ab́)2 and TFM and IgG, radiolabelled with both 131I and 90Y have been described previously using the human colorectal tumour LS174T nude mouse xenograft model (Casey et al (1996) Br J Cancer 74: 1397–1405). In this study quantitative estimates of radiation distribution and RIT in the xenograft model provided more insight into selecting the most suitable combination for future RIT. Radiation doses were significantly higher in all tissues when antibodies were labelled with 90Y. Major contributing organs were the kidneys, liver and spleen. The extremely high absorbed dose to the kidneys on injection of 90Y-labelled DFM and F(ab́)2 as a result of accumulation of the radiometal would result in extremely high toxicity. These combinations are clearly unsuitable for RIT. Cumulative dose of 90Y-TFM to the kidney was 3 times lower than the divalent forms but still twice as high as for 90Y-IgG. TFM clears faster from the blood than IgG, producing higher tumour to blood ratios. Therefore when considering only the tumour to blood ratios of the total absorbed dose, the data suggests that TFM would be the most suitable candidate. However, when corrected for equitoxic blood levels, doses to normal tissues for TFM were approximately twice the level of IgG, producing a two-fold increase in the overall tumour to normal tissue ratio. In addition RIT revealed that for a similar level of toxicity and half the administered activity, 90Y-IgG produced a greater therapeutic response. This suggests that the most promising A5B7 antibody form with the radionuclide 90Y may be IgG. Dosimetry analysis revealed that the tumour to normal tissue ratios were greater for all 131I-labelled antibodies. This suggests that 131I may be a more suitable radionuclide for RIT, in terms of lower toxicity to normal tissues. The highest tumour to blood dose and tumour to normal tissue ratio at equitoxic blood levels was 131I-labelled DFM, suggesting that 131I-DFM may be best combination of antibody and radionuclide for A5B7. The dosimetry estimates were in agreement with RIT results in that twice the activity of 131I-DFM must be administered to produce a similar therapeutic effect as 131I-TFM. The toxicity in this therapy experiment was minimal and further experiments at higher doses are required to observe if there would be any advantage of a higher initial dose rate for 131I-DFM. © 1999 Cancer Research Campaig

BAMBI Regulates Angiogenesis and Endothelial Homeostasis through Modulation of Alternative TGFβ Signaling

BACKGROUND: BAMBI is a type I TGFβ receptor antagonist, whose in vivo function remains unclear, as BAMBI(-/-) mice lack an obvious phenotype. METHODOLOGY/PRINCIPAL FINDINGS: Identifying BAMBI's functions requires identification of cell-specific expression of BAMBI. By immunohistology we found BAMBI expression restricted to endothelial cells and by electron microscopy BAMBI(-/-) mice showed prominent and swollen endothelial cells in myocardial and glomerular capillaries. In endothelial cells over-expression of BAMBI reduced, whereas knock-down enhanced capillary growth and migration in response to TGFβ. In vivo angiogenesis was enhanced in matrigel implants and in glomerular hypertrophy after unilateral nephrectomy in BAMBI(-/-) compared to BAMBI(+/+) mice consistent with an endothelial phenotype for BAMBI(-/-) mice. BAMBI's mechanism of action in endothelial cells was examined by canonical and alternative TGFβ signaling in HUVEC with over-expression or knock-down of BAMBI. BAMBI knockdown enhanced basal and TGFβ stimulated SMAD1/5 and ERK1/2 phosphorylation, while over-expression prevented both. CONCLUSIONS/SIGNIFICANCE: Thus we provide a first description of a vascular phenotype for BAMBI(-/-) mice, and provide in vitro and in vivo evidence that BAMBI contributes to endothelial and vascular homeostasis. Further, we demonstrate that in endothelial cells BAMBI interferes with alternative TGFβ signaling, most likely through the ALK 1 receptor, which may explain the phenotype observed in BAMBI(-/-) mice. This newly described role for BAMBI in regulating endothelial function has potential implications for understanding and treating vascular disease and tumor neo-angiogenesis

Proteomic characterization of HIV-modulated membrane receptors, kinases and signaling proteins involved in novel angiogenic pathways

<p>Abstract</p> <p>Background</p> <p>Kaposi's sarcoma (KS), hemangioma, and other angioproliferative diseases are highly prevalent in HIV-infected individuals. While KS is etiologically linked to the human herpesvirus-8 (HHV8) infection, HIV-patients without HHV-8 and those infected with unrelated viruses also develop angiopathies. Further, HIV-Tat can activate protein-tyrosine-kinase (PTK-activity) of the vascular endothelial growth factor receptor involved in stimulating angiogenic processes. However, Tat by itself or HHV8-genes alone cannot induce angiogenesis <it>in vivo </it>unless specific proteins/enzymes are produced synchronously by different cell-types. We therefore tested a hypothesis that <it>chronic </it>HIV-<it>replication in non-endothelial cells </it>may produce novel factors that provoke angiogenic pathways.</p> <p>Methods</p> <p>Genome-wide proteins from HIV-infected and uninfected T-lymphocytes were tested by subtractive proteomics analyses at various stages of virus and cell growth <it>in vitro </it>over a period of two years. Several thousand differentially regulated proteins were identified by mass spectrometry (MS) and >200 proteins were confirmed in multiple gels. Each protein was scrutinized extensively by protein-interaction-pathways, bioinformatics, and statistical analyses.</p> <p>Results</p> <p>By functional categorization, 31 proteins were identified to be associated with various signaling events involved in angiogenesis. 88% proteins were located in the plasma membrane or extracellular matrix and >90% were found to be essential for regeneration, neovascularization and angiogenic processes during embryonic development.</p> <p>Conclusion</p> <p>Chronic HIV-infection of T-cells produces membrane receptor-PTKs, serine-threonine kinases, growth factors, adhesion molecules and many diffusible signaling proteins that have not been previously reported in HIV-infected cells. Each protein has been associated with endothelial cell-growth, morphogenesis, sprouting, microvessel-formation and other biological processes involved in angiogenesis (p = 10^-4to 10^-12). Bioinformatics analyses suggest that overproduction of PTKs and other kinases in HIV-infected cells has <it>suppressed </it>VEGF/VEGFR-PTK expression and promoted <it>VEGFR-independent </it>pathways. This unique mechanism is similar to that observed in neovascularization and angiogenesis during embryogenesis. Validation of clinically relevant proteins by gene-silencing and translational studies <it>in vivo </it>would identify specific targets that can be used for early diagnosis of angiogenic disorders and future development of inhibitors of angiopathies. This is the first comprehensive study to demonstrate that HIV-infection alone, without any co-infection or treatment, can induce numerous "embryonic" proteins and kinases capable of generating novel <it>VEGF-independent </it>angiogenic pathways.</p

Biochemical­­– and biophysical–induced barriergenesis in the blood brain barrier: a review of barriergenic factors for use in in vitro models

Central nervous system (CNS) pathologies are a prevalent problem in aging populations, creating a need to understand the underlying events in these diseases and develop efficient CNS‐targeting drugs. The importance of the blood‐brain barrier (BBB) has become evident, acting both as a physical barrier to drug entry into the CNS, and potentially as the cause or aggravator of CNS diseases. The development of a biomimetic BBB in vitro model is required for the understanding of BBB‐related pathologies and in the screening of drugs targeting the CNS. There is currently a great interest in understanding the influence of biochemical and biophysical factors, as these have the potential to greatly improve the barrier function of brain microvascular endothelial cells (BMECs). Recent advances in understanding how these may regulate barriergenesis in BMECs can help promote the development of improved BBB in vitro models, and therefore novel interventional therapies for pathologies related to its disruption. This review provides an overview of specific biochemical and biomechanical cues in the formation of the BBB, with a focus on in vitro models and how these might recapitulate BBB function

Hypoxic Pulmonary Vasoconstriction in Humans:Tale or Myth

Hypoxic Pulmonary vasoconstriction (HPV) describes the physiological adaptive process of lungs to preserves systemic oxygenation. It has clinical implications in the development of pulmonary hypertension which impacts on outcomes of patients undergoing cardiothoracic surgery. This review examines both acute and chronic hypoxic vasoconstriction focusing on the distinct clinical implications and highlights the role of calcium and mitochondria in acute versus the role of reactive oxygen species and Rho GTPases in chronic HPV. Furthermore it identifies gaps of knowledge and need for further research in humans to clearly define this phenomenon and the underlying mechanism