Cell-oriented modeling of angiogenesis.

Due to its significant involvement in various physiological and pathological conditions, angiogenesis (the development of new blood vessels from an existing vasculature) represents an important area of the actual biological research and a field in which mathematical modeling proved particularly useful in supporting the experimental work. In this paper, we focus on a specific modeling strategy, known as "cell-centered" approach. This type of mathematical models work at a "mesoscopic scale," assuming the cell as the natural level of abstraction for computational modeling of development. They treat cells phenomenologically, considering their essential behaviors to study how tissue structure and organization emerge from the collective dynamics of multiple cells. The main contributions of the cell-oriented approach to the study of the angiogenic process will be described. From one side, they have generated "basic science understanding" about the process of capillary assembly during development, growth, and pathology. On the other side, models were also developed supporting "applied biomedical research" for the purpose of identifying new therapeutic targets and clinically relevant approaches for either inhibiting or stimulating angiogenesis.

Anatomical basis of hypoxic and hyperoxic injuries to the centres of cardiorespiratory regulation.

The aim of the present paper is to briefly review the changes occurring in the nucleus tractus solitarii and carotid body in response to hypoxic and hyperoxic injuries. Selective alterations of dendrites and Fos-immunoreactivity of neurons have been observed in the subnucleus gelatinosus of the nucleus tractus solitarii of adult subjects dying after hypoxic-ischaemic injury. The selective vulnerability of this portion of the nucleus tractus solitarii may be explained mainly with reference to the vascularization of medullary tegmentum. In the carotid body, chronic hypoxia and hyperoxia cause a series of morphological, cellular and biochemical changes which may play a major role during the first postnatal period and may have implications in the pathogenesis of Sudden Infant Death Syndrome. Intermittent hypoxia may cause hypersensitivity of the carotid body, possibly increasing the risk of unstable respiration. Conversely, hyperoxia exposure has been reported to cause hyposensitivity and reduction in volume of the carotid body, possibly leading to ineffective response.

The pro-angiogenic activity of urotensin-II on human vascular endothelial cells involves ERK1/2 and PI3K signaling pathways.

Human vascular endothelial cells express the urotensin-II (U-II) receptor and exhibit a strong in vitro angiogenic response to the peptide. Thus, in the present study an in vitro model, based on human umbilical vein endothelial cells (HUVEC) cultured on Matrigel, was used to characterize more in detail the signaling pathways that control the pro-angiogenic action of U-II. The activation of the U-II receptor (UT) was associated with an increase of intracellular calcium concentration. Both calcium rise and pro-angiogenic effect of the peptide can be blocked by U73122, a selective inhibitor of phospholipase-C, indicating that the signal transduction from UT mainly involves the phospholipase-C/IP(3) pathway. As far as the downstream signaling pathways are concerned, western blot analyses and experiments with specific inhibitors indicated that the U-II-induced self-organization of the cells into capillary-like structures was PKC dependent and involved the activation of the ERK1/2, but not p38-MAPK, transduction pathway. Interestingly, the pharmacological inhibition of PI3K (obtained with LY294002), hindered the capacity of U-II to induce a proangiogenic effect on HUVEC, suggesting that PI3K-dependent pathways also play a role in regulating the process.

Prolonged zidovudine administration induces a moderate increase in the growth and steroidogenic capacity of the rat adrenal cortex.

Zidovudine (AZT) is an antiretroviral drug widely used in the treatment of human immunodeficiency virus (HIV)-infected patients, whose prolonged administration was found to cause toxic lesions in cardiomyocytes in humans and experimental animals. Alterations in adrenocortical secretion were frequently observed in HIV patients, but it is not clear whether medication is involved in the production of these complications. Hence, we studied in vivo and in vitro, the effects of AZT on the rat adrenal cortex. The prolonged AZT administration (100 mg/kg per day for 4 months) did not cause overt qualitative morphological alterations of adrenocortical cells, which, however, underwent a net hypertrophy. Hypertrophy is associated with increases in the volume and surface area per cell of the mitochondrial compartment and smooth endoplasmic reticulum (where the enzymes of steroid synthesis are located), and a marked decrease in the volume of the lipid-droplet compartment (where cholesterol and its esters, the precursors of steroid hormones, are stored). AZT chronic treatment induced rises in the plasma concentrations of aldosterone and corticosterone, and in the basal and ACTH-stimulated in vitro secretion of these hormones from adrenal slices. The 24-h exposure to AZT (10(-5) M) did not significantly affect either secretory activity or proliferation and apoptotic rates of cultured rat adrenocortical cells. Taken together, these findings suggest that AZT chronic treatment enhances the growth and steroidogenic capacity of rat adrenal cortex, probably by activating the central branch of the hypothalamic-pituitary-adrenal axis. The toxic activity of AZT is thought to depend on increased production of ROS. On these grounds, it is likely that the lack of toxic effect of AZT on adrenocortical cells is due to their very elevated content in vitamin C, which prevents the deleterious effect of the AZT-induced increase in intracellular ROS production.

Up-regulation of adrenomedullin receptor gene expression in activated local stem cells during rat adrenal regeneration.

Previous studies showed that adrenomedullin (AM) gene expression was up-regulated in the regenerating rat adrenal cortex after enucleation and contra-lateral adrenalectomy, the effect being significant at day 1 after surgery and peaking between days 3 and 7. Using the same experimental model, we investigated by real time-polymerase chain reaction the mRNA expression of the AM receptor components: calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP)2 and 3. At time 0 (60 min after enucleation; control group), the CRLR mRNA content was approximately 2- and 5-fold higher than that of RAMP2 and RAMP3, respectively. No significant changes in CRLR mRNA expression were observed in relation to the time elapsed from enucleation. RAMP2 and RAMP3 mRNAs did not exhibit significant changes at day 1 after surgery, but underwent a marked increase between days 3 and 7. The mRNA content of the two RAMPs decreased at days 14 and 28, although remaining significantly higher than that of the controls. These findings indicate that the AM receptor subtypes AM1-R (CRLR-RAMP2) and AM2-R (CRLR-RAMP3) are up-regulated in enucleated adrenals, and the hypothesis is advanced that this effect depends on the increased local production of AM. The concerted increase in AM and its receptor expression would greatly improve the autocrine-paracrine mechanism(s) by which AM favors proliferation of zona glomerulosa stem cells during adrenal regeneration.

Up-regulation of adrenomedullin gene expression in the regenerating rat adrenal cortex.

Adrenomedullin (AM) is an endogenous regulatory peptide that exerts growth-promoting action in several normal and neoplastic tissues, and we investigated whether its gene expression changes during rat adrenal regeneration after enucleation and contra-lateral adrenalectomy. Regenerating adrenals were collected at day 0 (just after enucleation; control rats), 1, 3, 7, 14 and 28 after surgery. The immunocytochemical assay of PCNA (proliferating cell nuclear antigen) index confirmed that the early stages of regeneration can be divided into an initial differentiation period (from day 0 to day 3) and a subsequent high proliferative period (days 5 and 7) followed by a decrease in the proliferation activity. Real time-polymerase chain reaction (PCR) demonstrated that AM mRNA expression underwent a marked rise at day 1 of regeneration, attained a maximum level at days 3 and 5, and then declined from day 7, returning to the control value at days 14 and 28. Western blotting showed that AM protein expression was moderately elevated at day 1, was maximal between days 3 and 7, and then decreased at days 14 and 28, although remaining significant over the control value. Taken together, our findings indicate that the increase in the AM gene transcription and translation may be considered one of the early events in the enucleation-induced activation of local adrenocortical stem cells, conceivably favoring both the differentiation and proliferation stages of regeneration. The mechanism underlying this adrenocortical stem cell response does not seem to involve ACTH, because real time-PCR demonstrated that it also occurred in animals whose contralateral adrenal glands were spared, and consequently the level of circulating ACTH was in the normal range. It remains to be investigated whether the enucleation-induced relative hypoxia, ensuing from disruption of the vascular bed, and the local release of inflammatory cytokines may be involved in the up-regulation of AM gene expression in regenerating adrenal glands.

IGF-I enhances cortisol secretion from guinea-pig adrenal gland: in vivo and in vitro study.

Insulin-like growth factor (IGF)-I is a ubiquitously synthesized peptide that, along with IGF-II, acts via the IGF-R type I receptor. IGF-I and its receptor are expressed in the adrenal gland of humans and bovines, the secretion of which they seem to stimulate. As in humans and cows, the main glucocorticoid hormone secreted by guinea-pig adrenals is cortisol, and hence we have studied the adrenocortical effects of IGF-I in this species. In vivo experiments showed that prolonged IGF-I administration raised the plasma concentration of cortisol in both normal and dexamethasone/captopril-treated guinea pigs, thereby ruling out the possibility that IGF-I may act by activating the hypothalamic-pituitary-adrenal axis and the renin-angiotensin system. In vitro experiments demonstrated that IGF-I enhanced basal, but not maximally agonist [ACTH and angiotensin-II (Ang-II)]-stimulated, cortisol secretion from freshly dispersed guinea-pig inner adrenocortical cells. The IGF-I immuno-neutralization suppressed the IGF-I secretagogue effect, without altering the cortisol response to both ACTH and Ang-II. IGF-I raised cyclic-AMP and inositol triphosphate release from dispersed guinea-pig cells, and the effect was reversed by the adenylate cyclase inhibitor SQ-22536 and the phospholipase-C (PLC) inhibitor U-73122. SQ-22536, U-73122, the protein kinase (PK) A inhibitor H-89 and the PKC inhibitor calphostin-C decreased by approximately 50% the cortisol response of dispersed cells to IGF-I, and the combined exposure to SQ-22536 and U-73122 abolished it. We conclude that IGF-I stimulates glucocorticoid secretion from guinea-pig adrenocortical cells, acting via selective receptors coupled to both the adenylate cyclase/PKA- and PLC/PKC-dependent signaling cascades.

Galanin enhances corticosterone secretion from dispersed rat adrenocortical cells through the activation of GAL-R1 and GAL-R2 receptors coupled to the adenylate cyclase-dependent signaling cascade.

Galanin is a regulatory peptide, which acts via three subtypes of receptors, named GAL-R1, GAL-R2 and GAL-R3. Reverse transcription-polymerase chain reaction demonstrated the expression of GAL-R1 and GAL-R2, but not GAL-R3 mRNAs in dispersed rat adrenal zona fasciculata-reticularis (inner) cells. The immuno-blockade of GAL-R1 and GAL-R2, but not GAL-R3, decreased the binding of [3H]galanin to dispersed cells, a complete inhibition being obtained only by the simultaneous blockade of both receptor subtypes. Galanin stimulated corticosterone and cyclic-AMP release from dispersed inner rat adrenocortical cells, while inositol triphosphate production was not affected. Again these responses to galanin were reversed by both the GAL-R1 and GAL-R2, but not the GAL-R3 immuno-blockade. The adenylate cyclase inhibitor SQ-22536 and the protein kinase (PK) A inhibitor H-89 abolished the corticosterone response of dispersed cells to galanin, while the phospholipase C inhibitor U-73122 and the PKC inhibitor calphostin-C were ineffective. We conclude that rat inner adrenocortical cells express GAL-R1 and GAL-R2 as mRNA and protein, and galanin stimulates corticosterone secretion acting via these receptor subtypes which are both coupled to the adenylate cyclase/PKA-dependent signaling pathway.

Ouabain chronic infusion enhances the growth and steroidogenic capacity of rat adrenal zona glomerulosa: the possible involvement of the endothelin system.

Ouabain, an inhibitor of the Na+/K+-ATPase, has been reported to affect the secretory activity of the adrenal cortex, and especially of zona glomerulosa (ZG). However, conflicting results were obtained, depending on the experimental condition used since ouabain appears to interact with angiotensin-II (Ang-II) and its action to be influenced by the electrolyte balance. Hence, we investigated the effects of prolonged (4-month) infusion with ouabain on the rat adrenal cortex. Ouabain raised the plasma concentrations of aldosterone, corticosterone and endothelin-1 (ET-1), without affecting either systolic blood pressure (SBP) or plasma renin activity (PRA). The treatment caused a marked hypertrophy of ZG and ZG cells, which mainly ensued from increases in the volume of the mitochondrial and smooth-endoplasmic-reticulum compartments, where the enzymes of steroid synthesis are located. Conversely, the volume of the lipid-droplet compartment, which stores cholesterol utilized in steroid-hormone production, underwent a striking decrease. Zona fasciculata and its parenchymal cells were not affected. Basal and maximally agonist (ACTH, Ang-II and ET-1)-stimulated in vitro mineralocorticoid secretion from adrenal slices was also notably enhanced by ouabain administration. Collectively, these findings indicate that prolonged treatment with ouabain selectively stimulates the growth and steroidogenic capacity of the rat adrenal ZG. The possibility that the activation of the renin-angiotensin system may be involved in this effect of ouabain is ruled out by the lack of significant changes in SBP and PRA. Instead, our results suggest the possible involvement of ET-1, the plasma level of which is elevated in ouabain-infused rats.

Effects of some endocrine disruptors on the secretory and proliferative activity of the regenerating rat adrenal cortex.

The effects of some endocrine disruptors that possess estrogen-like activity on the secretion and growth of regenerating rat adrenal cortex have been investigated in ovariectomized (OVX) and sham-OVX rats. As reference groups, dexamethasone (Dx)-administered sham-OVX and 17beta-estradiol-administered OVX animals were used. Dx, estradiol and endocrine disruptors were subcutaneously injected daily at a dose of 3 nmoles/100 g for 10 consecutive days after surgery, and adrenal enucleation was performed on day 5 of the experiment. Dx and genistein significantly decreased corticosterone plasma concentration (as measured by RIA) in sham-OVX rats with regenerating adrenals, while other disruptors (eusolex, procymidone, linurone, resveratrol, bisphenol-A and and silymarin) were ineffective. Mitotic index (as assayed by the stachmokinetic method with vincristine) was not changed by either Dx or disruptors. Estradiol significantly increased and genistein significantly lowered corticosterone blood level in OVX rats; similar effects were induced in the mitotic index of regenerating adrenals, but the changes were not significant. Eusolex increased the mitotic index, without altering the level of circulating corticosterone. Collectively, our findings allow us to conclude that, of the endocrine disruptors tested, only genistein is able to suppress the secretory activity of regenerating adrenal cortex, this Dx-like effect being apparently unrelated to its estrogen-like activity, and only eusolex enhances the proliferation rate of regenerating adrenal, the effect being conceivably connected with its estrogen-like activity.

Angiogenic response induced by acellular femoral matrix in vivo.

We investigated the angiogenic response induced by acellular femoral matrices implanted in vivo on to the chick embryo chorioallantoic membrane (CAM), a useful model for such investigation. The results showed that acellular matrices were able to induce a strong angiogenic response, comparable with that of fibroblast growth factor-2 (FGF-2), a well-known angiogenic cytokine. The angiogenic response was further increased when exogenous FGF-2 or transforming growth factor beta-1 (TGF-beta1) was added to the matrices and inhibited by the addition of anti-FGF-2 or anti-TGF-beta1 antibodies. The response may be considered to be dependent on a direct angiogenic effect exerted by the matrices, and also in part by the presence of FGF-2 and TGF-beta1 in the acellular matrices.

Arginin-vasopressin regulates proliferative activity of the regenerating rat adrenal cortex.

Enucleation-induced adrenal regeneration is a classic model to investigate adrenocortical proliferation in vivo, which is dependent not only on pituitary ACTH release, but also on various other neural and endocrine signals. Arginin-vasopressin (AVP), mainly acting via V1 receptors, regulates hypothalamic-hypophyseal-adrenal axis function, acting on both its central and peripheral branches. Here, we studied whether endogenous AVP system modulates rat adrenal regeneration. Reverse transcription-polymerase chain reaction (PCR) detected only the mRNAs of V1a and V1b receptors in normal and regenerating adrenals. The expression was very low, and semi-quantitative conventional and real-time PCR showed that it was down-regulated in regenerating adrenals in relation to the time elapsed from enucleation. AVP (three subcutaneous injections 28, 16 and 4 h before sacrifice) raised metaphase index at day 5, but not at day 8 of regeneration. Unexpectedly, both V1-receptor and V2-receptor antagonists increased metaphase index at days 5 and 8 of regeneration. Neither AVP nor AVP-receptor antagonists affected plasma levels of corticosterone in rats bearing regenerating adrenals. It is concluded that AVP, acting via V1 receptors located in adrenals, exerts a stimulating effects on adrenal regeneration. Due to the down-regulation of V1-receptor expression in regenerating adrenals, this effect is very weak and is easily overcome by a tonic inhibitory action of endogenous AVP systems probably involving extra-adrenal indirect mechanisms.

Effects of met-enkephalin on cell proliferation in different models of adrenocortical-cell growth.

Met-enkephalin (met-Enk) is an opioid peptide that acts via three main subtypes of receptors referred to as mu (mu)-, delta (delta)- and zeta (zeta)-receptor. While the first two receptor subtypes mediate the classic opioid effects of met-Enk, zeta-receptors are reported to be involved in the non-opioid actions of the peptide, i.e. the inhibitory effect on the cell growth. Despite the fact that met-Enk is known to regulate the function of the hypothalamic-pituitary-adrenal axis acting on both its central and peripheral branches, none is known on the effects of met-Enk on adrenal growth. Hence, we have investigated the effects of met-Enk and its receptor agonists and antagonists on cell proliferation in three different models of rat adrenal growth: i) immature adrenal cortex, ii) regenerating adrenal cortex and iii) primary cultures of adrenocortical cells. In in vivo experiments, rats were given subcutaneous injections of 1 nmol/100 g of the peptides 28, 16 and 4 h before the sacrifice, and proliferative activity was assessed by counting the number of metaphase-arrested cells (after vincristine administration). In in vitro studies, cultured adrenocortical cells were exposed for 48 h to the peptides at a concentration of 10(-6) M, and proliferative activity was measured by the EZ4U method. The blockade of mu- and delta-receptors raised proliferative activity in immature adrenals and decreased it in regenerating glands, and the effects were reversed by mu- and delta-receptor agonists. Naltrexone-induced blockade of all met-Enk receptor subtypes decreased proliferative activity in immature adrenal and raised it in regenerating glands. The exposure to either mu- or delta-receptor agonists and antagonists evoked doubtful or no effects on the proliferative activity of cultured adrenocortical cells. In contrast, met-Enk exerted a marked antiproliferogenic effect that was reversed by naltrexone. Taken together, these findings allow us to draw the following conclusions: i) mu- and delta-receptor activation inhibits the growth of immature adrenals, stimulates adrenal regeneration, and does not affect proliferation of cultured adrenocortical cells; ii) zeta-receptors mediate the growth inhibitory effect of met-Enk on both regenerating adrenals and cultured adrenocortical cells, but unexpectedly their activation stimulates the growth of immature gland; and iii) the effects of mu- and delta-receptor activation in in vivo experiments are probably mediated by extra-adrenal indirect mechanisms.

Orexins modulate the growth of cultured rat adrenocortical cells, acting through type 1 and type 2 receptors coupled to the MAPK p42/p44- and p38-dependent cascades.

Orexin A and B are hypothalamic peptides that act through two subtypes of receptors named OX1-R and OX2-R. The OX1-R almost exclusively binds orexin-A, whereas OX2-R is non-selective for both orexins. We previously found that rat adrenocortical cells express both orexin-receptor subtypes, and orexin-A stimulates corticosterone secretion from dispersed adrenocortical cells acting via the OX1-R. Here, we examined the possibility that orexins, acting through both their receptor subtypes, modulate the growth of adrenocortical cells. Reverse transcription-polymerase chain reaction showed that rat adrenocortical cells cultured in vitro for four days expressed OX1-R and OX2-R mRNAs. Orexin-A increased the proliferation rate (PR) of cultured cells, while orexin-B lowered it. Using selective antibodies, we demonstrated that OX1-R immuno-blockade reversed the proliferogenic action of orexin-A, causing a sizeable decrease in PR. In contrast, OX2-R immuno-blockade magnified the proliferogenic effect of orexin-A and annulled the antiproliferogenic action of orexin-B. The proliferogenic effect of orexin-A in the presence of OX2-R immuno-blockade was abrogated by the MAPK p42/p44 inhibitor PD-98059, while the antiproliferogenic effect of orexin-A in the presence of OX1-R immuno-blockade was annulled by the MAPK p38 inhibitor SB-203580. Neither inhibitor altered per se the basal PR of cultured cells. Taken together, our present findings allow us to conclude that i) orexins modulate the growth of rat adrenocortical cells cultured in vitro, by exerting both proliferogenic and antiproliferogenic effects, which are mediated by OX1-Rs and OX2-Rs, respectively; and ii) OX1-R and OX2-R growth effects involve the activation of the MAPK p42/p44 and p38 signaling cascades, respectively.

Ghrelin inhibits FGF-2-mediated angiogenesis in vitro and in vivo.

Recent evidence indicates that ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), is highly expressed in the cardiovascular system, and in this study we addressed the possibility that ghrelin may affect angiogenesis in vitro and in vivo. Reverse transcription-polymerase chain reaction showed that human umbilical vein endothelial cells (HUVECs) express ghrelin and GHS-R mRNAs. Ghrelin inhibited FGF-2-induced proliferation of HUVECs cultured in vitro, the maximal effective concentration being 10(-8) M, and this effect was annulled by the GHS-R antagonist D-Lys3-growth hormone releasing peptide-6. FGF-2 stimulated HUVEC cultured on Matrigel to form capillary-like structures, and ghrelin (10(-8) M) suppressed this effect. In the chick embryo chorioallantoic membrane in vivo assay, FGF-2 induced a strong angiogenic response, which was counteracted by ghrelin (500 ng). Taken together, these findings suggest that ghrelin acts as an angiostatic molecule and indicate that its activity is comparable to that of a well-known angiostatic agent, i.e., vinblastine. The antiangiogenic activity of ghrelin deserves further investigations, alone or together with other antiangiogenic agents, for the treatment of pathological conditions characterized by enhanced angiogenesis.

Adrenomedullin (AM) and AM receptor type 2 expression is up-regulated in prostate carcinomas (PC), and AM stimulates in vitro growth of a PC-derived cell line by enhancing proliferation and decreasing apoptosis rates.

Adrenomedullin (AM) is a hypotensive peptide, that acts via the calcitonin receptor-like receptor (CRLR), whose interaction with the subtypes 2 and 3 of a family of receptor activity-modifying proteins (RAMP) gives rise to two distinct AM receptors, named AM1 and AM2 receptors. AM derives from the post-translational proteolytic cleavage of pro(p)AM, the last step of which involves the conversion of the inactive AM to active AM by the peptidyl-glycine alpha-amidating monooxigenase (PAM). Compelling evidence suggests that AM, in addition to exerting its well-known regulatory action on blood pressure and water and electrolyte balance, also possesses a growth promoting effect in several normal and neoplastic tissues, including human prostate. Conventional reverse transcription (RT)-polymerase chain reaction (PCR) demonstrated the expression of pAM, PAM, CRLR and RAMP(1-3) mRNAs in both prostate hyperplasias (PH) and carcinomas (PC), and semiquantitative PCR showed that pAM, PAM and RAMP3 mRNA expression was higher in PCs than PHs. Radioimmunoassay measured higher concentrations of immunoreactive AM in PCs than PHs. The expression of pAM, CRLR and RAMP1,2 mRNAs was also detected in the PC-derived cell lines PC-3 and DU-145, RAMP3 expression being restricted to the latter line. AM did not affect the growth rate (duplication time) of PC-3 cells, but it did significantly increase that of DU-145 cells. The growth promoting effect of AM was found to ensue from both the rise in the proliferation rate and the lowering in the apoptosis rate of DU-145 cells. These effects of AM were counteracted by the AM receptor antagonists CGRP(8-37) and AM(22-52), the former antagonist, which is more selective for AM2 than AM1 receptors, being more effective than the latter one. Both antagonists were per se able to induce a slow, but significant decrease in the basal growth rate of DU-145 cells by inhibiting proliferation and enhancing apoptosis, again CGRP(8-37) being more effective than AM(22-52). Taken together, our findings allow us to suggest that: i) endogenous AM system plays an important autocrine-paracrine growth promoting action in the human prostate, being possibly involved in the development of the malignant phenotype of epithelial cells; and ii) the tumor promoting effect of AM in the human prostate is mainly mediated by the AM2 receptor (CRLR/RAMP3) subtype.

In vitro culture of rat neuromicrovascular endothelial cells on polymeric scaffolds.

Polyphosphazenes are polymers possessing a skeleton composed of alternating phosphorous and nitrogen atoms, and two side-moieties linked to each phosphorous atom. Polyphosphazenes with amino acid esters as side-moieties are biocompatible and biodegradable polymers. Two polyphosphazenes, poly[bis(ethyl alanate) phosphazene] and poly[(ethyl phenylalanate)0.8(ethyl alanate)0.8(ethyl glycinate)0.4 phosphazene] (PPAGP) were synthesized, and processed to form small fibers. Their ability to support rat neuromicrovascular endothelial cell (EC) adhesion and growth has been studied, using poly(D,L-lactic acid) as reference compound. Scanning electron microscopy revealed that both poly[bis(ethyl alanate) phosphazene] and PPAGP fibers were thinner than poly(D,L-lactic acid) fibers, and possessed a more irregular and porous surface. All polymers increased EC adhesion, compared with polystyrene, but only polyphosphazenes were able to improve EC growth. The highest increase in EC proliferation was induced by PPAGP, which, as revealed by environmental scanning electron microscopy, was also able to induce ECs to arrange into tubular structures. The conclusion is drawn that PPAGP may provide the best scaffold for engineered blood vessels, because it promotes adhesion, growth, and organization of ECs into capillary-like structures.

Ghrelin enhances the growth of cultured human adrenal zona glomerulosa cells by exerting MAPK-mediated proliferogenic and antiapoptotic effects.

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), two subtypes of which have been identified and named GHS-R1a and GHS-R1b. Evidence has been provided that ghrelin and its receptors are expressed in the adrenal gland, and we have investigated the possible role of the ghrelin system in the functional regulation of the human adrenal cortex. Reverse transcription-polymerase chain reaction detected the expression of both subtypes of GHS-Rs exclusively in the zona glomerulosa (ZG). Ghrelin did not significantly affect either basal or agonist-stimulated aldosterone secretion from cultured ZG cells. In contrast, ghrelin raised proliferative activity and decreased apoptotic deletion rate of ZG cells, the maximal effective concentration being 10(-8) M. The growth effects of 10(-8) M ghrelin on cultured ZG cells were not affected by either the protein kinase (PK)A and PKC antagonists H-89 and calphostin-C or the mitogen-activated PK (MAPK) p38 antagonist SB-293580, but were abolished by both the tyrosine kinase (TK) and MAPK p42/p44 antagonists tyrphostin-23 (10(-5) M) and PD-98059 (10(-4) M), respectively. Ghrelin (10(-8) M) enhanced TK and MAPK p42/p44 activities of ZG cells. Preincubation with 10(-5) M tyrphostin-23 blocked the ghrelin-induced stimulation of both TK and MAPK p42/p44, while preincubation with 10(-4) M PD-98059 only annulled MAPK p42/p44 stimulation. Collectively, our findings allow us to conclude that ghrelin, acting via GHS-Rs exclusively located in the ZG, enhances the growth of human adrenal cortex, through a mechanism involving the activation of the TK-dependent MAPK p42/p44 cascade.

Beacon[47-73] inhibits glucocorticoid secretion and growth of cultured rat and human adrenocortical cells.

Evidence has been recently provided that beacon, an ubiquitin-like protein overexpressed in the hypothalamus of Israeli sand rat, is also expressed in several endocrine glands of the Wistar rat, including adrenal cortex. Moreover, it has been shown that the in vivo administration of beacon[47-73] (hereinafter, beacon) evokes within 60 min a marked decrease in the plasma concentrations of ACTH and corticosterone. Hence, we have investigated the effect of beacon (4x10(-9) or 4x10(-7) M) on the secretion and growth of cultured rat and human zona fasciculata/reticularis (ZF/R) cells. Reverse transcription-polymerase chain reaction detected beacon mRNA in all human adrenal cortexes examined. A 3-h exposure to beacon was ineffective, but prolonged (24 and 96 h) exposures significantly lowered basal corticosterone and cortisol secretion from cultured rat and human ZF/R cells, respectively. Moreover, beacon (4x10(-7) M) counteracted the secretagogue action of 10(-8) M ACTH on cultured cells. The 96-h exposure to beacon concentration-dependently decreased basal proliferation rate of cultured cells, without inducing significant changes in the number of apoptotic and necrotic cells. Beacon (4x10(-7) M) significantly inhibited the proliferogenic effect of 10(-8) M adrenomedullin. In light of the involvement of ubiquitin-like proteins in the control of cell cycle and protein sorting and degradation, the hypothesis is advanced that the inhibitory effect of beacon on the secretion and growth of cultured rat ZF/R cells may be connected to its stimulating effect on proteolysis of steroidogenic enzymes and proteins involved in cell replication.

Effect of ghrelin on the apoptotic deletion rate of different types of cells cultured in vitro.

Evidence indicates that ghrelin, an endogenous ligand of the growth hormone secretagogue receptor, controls the growth of several human and rat cell types cultured in vitro. Hence, we have investigated, by using both TUNEL and ELISA assays, the effects of 10(-8) M ghrelin on the basal apoptotic deletion rate of rat osteoblasts and thymocytes, rat and human adrenocortical cells, human umbilical vein endothelial cells, and human aldosteronoma cells cultured in vitro, as well as of the human adrenocortical carcinoma-derived cell lines NCI-H295 and SW-13. Both assays consistently showed that ghrelin did not affect apoptotic rate of normal rat and human cells, but significantly enhanced apoptotic deletion in aldosteronoma, NCI-H295 and SW-13 cell cultures. Due to the central role of apoptosis in the control of tumor growth, these findings, if confirmed in other tumor cell types, could suggest an antitumoral action of ghrelin.