Functional significance of hsp90 complexes with NOS and sGC in endothelial cells.

Although the existence of hsp90-NOS and hsp90-sGC complexes is now firmly established, their role in many pathophysiological processes remain unclear. These complexes may represent physiological mechanisms aimed at maximizing intracellular cGMP production in response to endogenous or drug-derived NO in endothelial cells and thus affecting permeability, proliferation, migration and apoptosis. Along with minimizing NO scavenging by superoxide and reducing the formation of peroxynitrite, these complexes may also prolong sGC stability by retarding its degradation. Our work and that of others have demonstrated that, depending on the environment, sGC interaction with hsp90 can optimize sGC enzyme activity or modulate sGC survival. This review addresses the functional significance of hsp90 complexes with NOS (eNOS, iNOS) and sGC in endothelial cells relevant for maintaining endothelial barrier integrity and angiogenesis. Structural and functional characteristics of sGC, its expression, transcriptional and post-translational regulation, as they relate to sGC-hsp90 interactions, will also be examined.

Effect of local anti-VEGF antibody treatment on tumor microvessel permeability.

Recent studies have shown that systemic injection of anti-VEGF antibody into tumor-bearing mice results in decreases in tumor vascular permeability, vessel diameters, and tumor regression. Using a similar animal model, we have applied anti-VEGF antibody directly to the tumor tissue growing in transparent window chambers in SCID mice. Similar to the results obtained with systemic injection, vascular permeability was greatly reduced, but the response was reached at much lower concentrations with local application. Implications of these findings on local control of tumors are discussed.

Angiogenic potential of malignant and non-malignant human breast tissues in an in vivo angiogenesis model.

Tumors need to acquire an angiogenic phenotype for outgrowth and metastasis formation. Limited information on the angiogenic potential of specific tissues, especially human breast tissues is available. Here we describe an in vivo model, using the dorsal skin fold chamber in immunodeficient nude mice, where various tissues of human breast origin were xenografted and evaluated for their angiogenesis-inducing potential. We found that angiogenesis was abundantly induced by all breast carcinoma tissue samples. Similar angiogenesis was induced by tissue samples from breasts with hyperplasia and apocrine metaplasia. Histologically normal tissues adjacent to the tumor induced angiogenesis in 66% of the cases. Angiogenesis was not induced by control tissues from normal healthy breasts, obtained after cosmetic breast reduction. Angiogenesis induction parallelled VEGF production by the tumor cells. The tissue induced neovascularization, found both around and in the human tissue, was functional since a tail vein injection of albumin-FITC revealed positive tumor microcirculation within 5 min, while the tumor tissue still consisted of vital human epithelial cells after 14 days.

Cellular arrangement of human breast cancer cell lines determines hemostatic parameters.

Two in vitro models are compared to investigate whether cellular configuration or composition of the matrix in which the cells are cultured influences growth and/or prognostic parameters. T47D, MCF-7 and Hs578T breast cancer cell lines were cultured on two different matrices (agarose and collagen). Growth curves, biological markers (Ki-67, p53 and bcl-2) and the expression of hemostatic parameters were studied. The tested hemostatic parameters were urokinase-type plasminogen activator, tissue-type plasminogen activator and plasminogen activator inhibitor as fibrinolytic parameters and von Willbrand factor, tissue factor, antithrombin III, factor X and factor Xa as coagulation parameters. We found that T47D and MCF-7 formed spheroids in both matrices. Hs578T did not form spheroids; instead, the cells remained single cells in the agarose matrix and grew invasively through the collagen matrix. Expression of the biological markers was similar for spheroids and monolayers. In contrast, a clear difference in expression of hemostatic factors by spheroids and monolayers was found.

Solid stress inhibits the growth of multicellular tumor spheroids.

In normal tissues, the processes of growth, remodeling, and morphogenesis are tightly regulated by the stress field; conversely, stress may be generated by these processes. We demonstrate that solid stress inhibits tumor growth in vitro, regardless of host species, tissue of origin, or differentiation state. The inhibiting stress for multicellular tumor spheroid growth in agarose matrices was 45 to 120 mm Hg. This stress, which greatly exceeds blood pressure in tumor vessels, is sufficient to induce the collapse of vascular or lymphatic vessels in tumors in vivo and can explain impaired blood flow, poor lymphatic drainage, and suboptimal drug delivery previously reported in solid tumors. The stress-induced growth inhibition of plateau-phase spheroids was accompanied, at the cellular level, by decreased apoptosis with no significant changes in proliferation. A concomitant increase in the cellular packing density was observed, which may prevent cells from undergoing apoptosis via a cell-volume or cell-shape transduction mechanism. These results suggest that solid stress controls tumor growth at both the macroscopic and cellular levels, and thus influences tumor progression and delivery of therapeutic agents.

Perfusion of single tumor microvessels: application to vascular permeability measurement.

OBJECTIVE: To develop a new method for determining the relative importance of convection versus diffusion in macromolecular transport across tumor microvessel walls. METHODS: The human colon adenocarcinoma LS174T was transplanted in the dorsal skinfold chamber in a severe combined immunodeficient (SCID) mouse. The vasculature at the tumor surface was exposed by carefully removing the glass window of the chamber. A tumor microvessel was randomly selected, which was approximately 20-40 microns in diameter, embedded in the connective tissue 10-12 microns below the surface of the tumor. The vessel was cannulated with a micropipette and perfused with fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) at different perfusion pressures. The fluorescence intensity was recorded on videotapes via a video system attached to the fluorescence microscope for offline analysis. The apparent vascular permeability was determined based on the time-dependence of fluorescence intensity and the vessel diameter. RESULTS: The apparent vascular permeability of single vessels to FITC-labeled BSA was quantified at perfusion pressures of 20-45 cmH2O. The pressure dependence of vascular permeability in LS174T tumors was heterogeneous. On average, there was no correlation between the apparent vascular permeability and the perfusion pressure in the range of 20-35 cmH2O (p = 0.73), even though the apparent permeability increased significantly when the pressure was increased from 20 to 45 cmH2O (p = 0.008). CONCLUSIONS: These results indicate that convection in the transvascular transport of albumin is not significant in non-peripheral regions of solid tumors in which the pressure difference across the vessel wall is small or even negligible. In addition to the permeability studies, this preparation can be used to study cell-cell interactions in single tumor vessels under defined flow conditions.

Tumour angiogenesis: pathophysiology and clinical significance.

In the 1970s it was first proposed that tumours depended on the establishment of a microcirculation in order to grow beyond a few millimetres. Thereafter, the search to prove this hypothesis increased strongly and by the end of the 1980s, evidence was given that tumours were angiogenesis-dependent and metastatic cells were only shed after the tumour had established its microcirculation. The process of neovascularization is regulated by numerous growth factors, vascular endothelial cells, and matrix proteins released from host stromal cells such as macrophages and mast cells. The process of tumour growth and metastasis involves tumour cell-host cell and cell-matrix interactions and many of the underlying mechanisms of these interactions still remain to be elucidated. Although in a minority of cases treatment of solid tumours has been effectively improved, for the majority of cases more adequate treatment is still required to reduce the mortality rate. It has been proposed only recently that specific targeting of the angiogenic process might inhibit tumour growth and metastasis. This promising field of research is now exponentially growing. It is the purpose of this review to summarize current knowledge on the pathophysiology and clinical significance of tumour angiogenesis.

Tumor spheroid-induced vesicle formation on endothelial cells is associated with procoagulant properties.

Fibrin deposits in tumor beds are an intriguing phenomenon. It has been suggested that fibrin plays a role as a provisional matrix in which the tumor grows and induces development of a vascular network. On the other hand fibrin possibly protects the tumor nodule from host defense mechanisms. We therefore investigate whether tumor cells can induce a procoagulant response in endothelial cells leading to fibrin formation. For our studies we employed a modification of the matrix model of Montesano in which sprouting of endothelial cell aggregates can be followed. This system allows us to study in vitro the involvement of coagulation in tumor growth and angiogenesis. Cocultures of endothelial cell aggregates and avascular tumor spheroids in collagen type I gels results in the appearance of extracellular vesicle-like structures on the endothelial sprouts. The vesicles formed on endothelial cell sprouts upon coculturing with tumor cells exhibit an increased amidolytic activity, suggestive of factor X/Xa activity, not dependent on tissue factor exposure. Experiments using HgCl2 and Iodoacetamide point to the importance of SH groups in the factor X/Xa activity on endothelial cell sprouts.

Tumor cell spheroids induce a mitogenic response in endothelial cells.

Conditioned media from spheroids of human colorectal tumor cell lines HT29, SW620, SW480 and CaCo-2 were tested for their mitogenic activity on human umbilical vein endothelial (HUVE) and bovine capillary endothelial (BCE) cells. All spheroid-conditioned media were capable of increasing the labeling indices in both endothelial cell populations, with the mitogenic activity decreasing in the following order: SW620, HT29/SW480 and CaCo-2. The endothelial cell mitogenic activity of the spheroids was not dependent on the presence of necrosis nor on their in vivo cytokinetic characteristics. We conclude that tumor spheroids release mitogenic activity for endothelial cells, but this stimulation depends on the age of the tumor spheroid as well as on the type of tumor used.

Effect of maternal exposure to smoke on gas diffusion capacity in neonatal rat.

Offspring of control and experimental (chronically exposed to whole cigarette smoke) rats were sacrificed on 15th postnatal day. Pulmonary tissues were processed for quantitative electron microscopic analyses. Arithmetic and harmonic mean thicknesses were directly measured and diffusion capacity was derived for separate layers (tissue, plasma, erythrocyte) and the entire lung. Experimental offspring showed a greater volume density of interstitium in parenchyma (P less than 0.04), arithmetic (P less than 0.05) and harmonic (P less than 0.009) mean thicknesses. Diffusion capacities for lungs of both animal groups, however, were similar (0.00543 and 0.0054 cm3 O2.min-1.mm Hg-1.g body weight-1 for control and experimental lungs respectively). Experimental tissue appeared better adapted for optimal exchange of gases (P less than 0.05), as indicated by corrugation index (ratio between arithmetic and harmonic mean thicknesses) than for the control counterpart. In spite of induced alterations in several developmental processes by a presently used experimental procedure, tissue capacity for transport of gases was not significantly changed in either the alveolo-capillary membrane or the entire lung.