Role of TGFβ1 and WNT6 in FGF2 and BMP4-driven endothelial differentiation of murine embryonic stem cells

Embryonic stem cells (ES) are a valuable source of endothelial cells. By co-culturing ES cells with the stromal PA6 cells, the endothelial commitment can be achieved by adding exogenous FGF2 or BMP4. In this work, the molecular pathways that direct the differentiation of ES cells toward endothelium in response to FGF2 are evaluated and compared to those activated by BMP4. To this purpose the genes expression profiles of both ES/PA6 co-cultures and of pure cultures of PA6 cells were obtained by microarray technique at different time points. The bioinformatics processing of the data indicated TGFβ1 as the most represented upstream regulator in FGF2-induced endothelial commitment while WNT pathway as the most represented in BMP4-activated endothelial differentiation. Loss of function experiments were performed to validate the importance of TGFβ1 and WNT6 respectively in FGF2 and BMP4-induced endothelial differentiation. The loss of TGFβ1 expression significantly impaired the accomplishment of the endothelial commitment unless exogenous recombinant TGFβ1 was added to the culture medium. Similarly, silencing WNT6 expression partially affected the endothelial differentiation of the ES cells upon BMP4 stimulation. Such dysfunction was recovered by the addition of recombinant WNT6 to the culture medium. The ES/PA6 co-culture system recreates an in vitro complete microenvironment in which endothelial commitment is accomplished in response to alternative signals through different mechanisms. Given the importance of WNT and TGFβ1 in mediating the crosstalk between tumor and stromal cells this work adds new insights in the mechanism of tumor angiogenesis and of its possible inhibition.

     

Effect of short-term cyclosporine administration in rats on renin-angiotensin and thromboxane A2: possible relevance to the reduction in glomerular filtration rate

A short-term treatment with Cyclosporine A (CyA) induces a decrease in glomerular filtration rate (GFR), promptly reversible after withdrawal of the drug. Several lines of evidence are now available as to indicate that this phenomenon is dependent on a hemodynamic perturbation resulting in a renal vasoconstriction. With the present work we have examined the relationship between the reduction in GFR which follows a short-term administration of CyA in rats and the biochemical changes in renin-angiotensin system and renal arachidonic acid metabolism. Our results show that CyA administration (25 mg/kg/day) for 45 days stimulates renin-angiotensin system with an increase in plasma renin activity. These changes are not accompanied by a parallel increase in the renal synthesis of vasodilatory prostaglandin E2 and prostacyclin as it occurs in other conditions of renin-angiotensin stimulation. At variance glomerular synthesis and urinary excretion of thromboxane A2 (TxA2) are increased progressively during CyA treatment. These changes in renal Tx precede the increase in serum creatinine and the decrease in GFR thus indicating that TxA2 might be an additional factor potentiating the effect of angiotensin II on glomerular hemodynamics. In conclusion the early reduction in GFR which follows daily administration of CyA in rats might be the result of a synergic action of angiotensin II and TxA2 on vascular tone and mesangial contraction which is not modulated by an increase in glomerular vasodilatory prostaglandins. If this explanation may be applied to early reduction in GFR observed in humans treated with CyA before tubular toxicity develops needs to be investigated further.     

Alterations of blood vessel development by endothelial cells overexpressing fibroblast growth factor-2

A close relationship exists between angiogenesis and the formation of vascular lesions. The development of the vascular system in the chick embryo chorioallantoic membrane (CAM) may thus represent a model to study the effects of the deregulation of endothelial cell behaviour. Alterations of the developing vascular tree of the CAM were observed after exposure to murine aortic endothelial (MAE) cells overexpressing human fibroblast growth factor-2 (FGF2) cDNA (pZipFGF2 MAE cells), or to their conditioned medium (CM). pZipFGF2 MAE cells injected into the allantoic sac or applied on to the CAM of day 8-9 chick embryos induce neovascularization and the appearance of haemangioma-like lesions. This activity was not prevented by anti-FGF2 antibodies. The CM from pZipFGF2 MAE cells was also active when adsorbed into a gelatin sponge and applied on to the CAM, both in the absence and in the presence of anti-FGF2 antibodies. No effects on vessel development were exerted by parental MAE cells, FGF2-transfected NIH 3T3 fibroblasts, or their conditioned media. In vitro, pZipFGF2 MAE cell CM caused parental MAE cells to invade fibrin gels and to undergo morphogenesis on Matrigel. This activity was not mimicked by recombinant FGF2 nor affected by anti-FGF2 antibodies, and depended on a M (r) approximately 45 000 heat-labile heparin-binding factor. Size exclusion chromatography of pZipFGF2 MAE cell CM demonstrated that the in vitro activity co-purified with an in vivo angiogenic capacity. Thus, FGF2 overexpression in mouse endothelial cells induces the production of an angiogenic activity distinct from FGF2, which may contribute to the genesis of angioproliferative lesions.     

Angiogenic phenotype induced by basic fibroblast growth factor transfection in brain microvascular endothelial cells

Basic fibroblast growth factor (bFGF) is expressed in the vascular endothelium of human brain tumors. To investigate the biological consequences of a possible autocrine modality of microvascular endothelial cell activation by endogenous bFGF in these tumors, mouse brain microvascular endothelial cells were stably transfected with a retroviral expression vector harboring a human bFGF cDNA. When grown on tissue culture plastic, bFGF-transfected clones show a transformed morphology and increased saturation density. bFGF-transfectants have an invasive behavior when seeded on three-dimensional fibrin gel and originate endothelial cell sprouts when embedded within fibrin. Also, bFGF-transfected cells undergo morphogenetic organization and produce a complex network of branching cord-like structures connecting foci of infiltrating cells when seeded on Matrigel, a laminin-rich extracellular matrix material. In contrast, parental and mock-transfected cells do not invade fibrin gels nor organize on Matrigel. These findings demonstrate that bFGF overexpression induces an angiogenic phenotype in brain microvascular endothelial cells characterized by an invasive behavior and morphogenic potential. They support the notion that neovascularization of brain tumors can be triggered by stimuli that induce vascular endothelium to produce its own autocrine factor(s).     

Urinary albumin excretion in normal pregnancy and pregnancy-induced hypertension.

We measured the urinary excretion of albumin in 67 healthy primigravidae, at monthly intervals, from 16 to 36 weeks of gestation and 12 weeks postpartum. Of the 67 primigravidae, 55 completed a normal pregnancy and 12 developed pregnancy-induced hypertension. In the latter group, an additional measurement of urinary albumin excretion was performed at 24 weeks postpartum. The aims of the study were: to look for changes of urinary albumin excretion during the progression of normal pregnancy; to assess if microalbuminuria could be an early feature of pregnancy-induced hypertension; to evaluate the effects of physical activity on the excretion of albumin in normal pregnancy and pregnancy-induced hypertension. In contrast with glomerular hyperfiltration and increased urinary total protein, two recognized characteristics of the pregnant state, we found that normal primigravidae, during the day, excrete significantly less albumin (p between less than 0.01 and less than 0.001) in comparison with the postpartum period and nonpregnant women. Normal primigravidae, as a group, showed parallel changes of urinary albumin excretion and diastolic blood pressure throughout pregnancy and postpartum, suggesting an important physiologic role of hemodynamic factors in regulating glomerular permeability to albumin. The daytime urinary albumin excretion in patients developing pregnancy-induced hypertension was significantly higher (p between less than 0.005 and less than 0.001) than in normal pregnancy from the 28th gestational week onwards. The increased urinary albumin excretion preceded the onset of hypertension and tended to persist long after blood pressure had returned to normal levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Basic fibroblast growth factor in human pheochromocytoma: A biochemical and immunohistochemical study

Biopsies of human normal adrenal medulla, adrenal pheochromocytoma, and chemodectoma were studied for the presence of basic fibroblast growth factor (bFGF). An immunoreactive Mr 18,000 bFGF-like molecule was detected both in normal and neoplastic tissues. This molecule was identified as bFGF on the basis of its molecular weight, its affinity for heparin, and its capacity to induce plasminogen activator production in cultured endothelial GM 7373 cells. The levels of immunoreactive and biologically active bFGF appeared to be significantly higher in the extracts of adrenal pheochromocytoma and chemodectoma than in the extracts of normal adrenal medulla. bFGF immunostaining was detectable in the nuclei of chief (Type-I) cells and of endothelial cells both in normal adrenal medulla and in pheochromocytoma. Cytoplasmic bFGF positivity of endothelial cells was also observed in pheochromocytoma but not in normal tissue. The data are in keeping with the hypothesis that bFGF may exert autocrine and paracrine functions in the growth and neovascularization of human pheochromocytoma.     

Endothelial cells overexpressing basic fibroblast growth factor (FGF-2) induce vascular tumors in immunodeficient mice

Basic fibroblast growth factor (FGF-2) is expressed in vascular endothelium during tumor neovascularization and angioproliferative diseases, including vascular tumors and Kaposi's sarcoma (KS). We have investigated the in vivo biological consequences of endothelial cell activation by endogenous FGF-2 in a mouse aortic endothelial cell line transfected with a retroviral expression vector harboring a human FGF-2 cDNA and the neomycin resistance gene. FGF-2 transfectants, named pZipbFGF2-MAE cells, caused the rapid growth of highly vascularized, non-infiltrating tumors when injected in nude mice. In contrast, lesions grew poorly when cells were injected in immunocompetent syngeneic animals. Histologically, the tumors had the appearance of hemangioendothelioma with spindled areas resembling KS and with numerous CD31+ blood vessels and lacunae. Southern blot analysis of tumor DNA, as well as disaggregation of the lesion followed by in vitro cell culture, revealed that less than 10% of the cells in the tumor mass retain FGF-2 overexpression and neomycin resistance at 6–8 weeks post-injection. Nevertheless, in vitro G418 selection allowed the isolation from the tumor of a FGF-2-overexpressing cell population showing biochemical and biological characteristics similar to those of pZipbFGF2-MAE cells, including the capacity to originate vascular lesions when re-injected in nude mice. To evaluate the effect of angiostatic compounds on the growth and vascularization of pZipbFGF2-MAE cell-induced lesions, nude mice were treated weekly (100mg/kg, i.p.) with the angiostatic sulfonated distamycin A derivative 2,2-(carbonyl-bis-[imino-N-methyl-4,2-pyrrole carbonyl-imino-{N-methyl-4,2-pyrrole}carbonylimino])-bis-(1,5-naphthalene) disulfonic acid (PNU 153429). The results demonstrate that PNU 153429 inhibits the growth of the lesions and causes a 50% decrease in CD31+ microvessel density. In conclusion, the data indicate that FGF-2-overexpressing endothelial cells cause vascular lesions in immunodeficient mice which may represent a novel model for opportunistic vascular tumors suitable for the evaluation of angiostatic compounds.     

Hormone evaluation in brain death

BACKGROUND: In this study, the level and the variation of a number of hormone and metabolic parameters during brain death treatment in potential organ donors have been monitored. METHODS: Thirty-nine consecutive brain-dead patients were enrolled in 3 Intensive Care Units of Regional Hospitals of the North of Italy. All patients were potential organ donors and free from diseases before the accident leading to death. The levels of ADH, ACTH, TSH, prolactin, cortisol, aldosterone, FT3, FT4, renin, serum lactate and plasma osmolality were measured immediately after the diagnosis of brain death (T0), certified following the Italian law of December 29, 1993, n. 578, and after 6 hours (T6). RESULTS: Hormone levels were normal in the majority of subjects, and there was no significant variation during the 6 hours of the observation period. No correlation was found between the hormone levels considered and the metabolic parameters; ADH levels were not correlated with plasma osmolality. FT3 levels were below the normal range in the majority of subjects, but were not associated with a higher lactate level, which is used as a marker of a shift toward tissue anaerobic metabolism. CONCLUSIONS: In conclusion, triiodothyronine administration to improve metabolic order and thus the function of organs for transplantation is not justified in brain-dead patients.