Hereditary Hemorrhagic Telangiectasia (HHT), also known as Rendu-Osler syndrome, is a genetic vascular disorder affecting 1 in 5000–8000 individuals worldwide. This rare disease is characterized by various vascular defects including epistaxis, blood vessel dilations (telangiectasia) and arteriovenous malformations (AVM) in several organs. About 90% of the cases are associated with heterozygous mutations of ACVRL1 or ENG genes, that respectively encode a bone morphogenetic protein receptor (activin receptor-like kinase 1, ALK1) and a co-receptor named endoglin. Less frequent mutations found in the remaining 10% of patients also affect the gene SMAD4 which is part of the transcriptional complex directly activated by this pathway. Presently, the therapeutic treatments for HHT are intended to reduce the symptoms of the disease. However, recent progress has been made using drugs that target VEGF (vascular endothelial growth factor) and the angiogenic pathway with the use of bevacizumab (anti-VEGF antibody). Furthermore, several exciting high-throughput screenings and preclinical studies have identified new molecular targets directly related to the signaling pathways affected in the disease. These include FKBP12, PI3-kinase and angiopoietin-2. This review aims at reporting these recent developments that should soon allow a better care of HHT patients.
Endothelial cells lining vessels of endocrine tissues are fenestrated. Interactions with the local environment via either soluble factors or cell-cell interactions appear to govern this terminal endothelial differentiation. Adrenocorticotropin (ACTH) has previously been reported to modulate endothelial fenestration in the rat adrenal cortex. Since vascular endothelial growth factor (VEGF) has been characterized as a potent inducer of endothelial fenestration, we aimed to characterize the status of VEGF expression in the bovine adult adrenal cortex and asked whether ACTH may regulate VEGF expression. By immunohistochemical analysis, we observed VEGF expression in steroidogenic cells from both zona glomerulosa and zona fasciculata of the bovine adrenal cortex. Double-labeling experiments performed on isolated cells in primary culture revealed VEGF immunoreactivity, essentially colocalized with the Golgi apparatus. The expression of two predominant VEGF isoforms, VEGF(121) and VEGF(165), was observed by RT-PCR analysis. ACTH (10 nM) was found to rapidly (within 2-4 h) increase the abundance of these VEGF transcripts, as assessed by both RT-PCR and Northern blot analysis. In parallel, ACTH significantly induced VEGF secretion into the medium of fasciculata cells in primary culture. Thus, our data are consistent with the involvement of ACTH, through its regulation of VEGF expression, in the maintenance of the adult adrenal cortex endothelium.
The extracellular matrix (ECM) strongly contributes to the regulation of cell proliferation and cell differentiation, and thereby of embryonic development and adult tissue homeostasis. We review here the ongoing characterization of the structure and functions of the extracellular matrix components secreted by adrenocortical cells and discuss their possible implication in the hormonal regulation of adrenal cortex homeostasis. Fibronectin (FN) and laminin (LN) are both major adhesive proteins for adrenocortical cells. FN is synthesized by bovine fasciculata cells in primary culture, and its synthesis is stimulated by TGF(beta)1, TGF(beta)2, and FGF-2 but is not modified by IGF-1 or by the hormones ACTH and angiotensin II. LN is also synthesized by bovine fasciculata cells and its synthesis is specifically stimulated by ACTH. Both proteins are haptotactic and chemotactic for adrenocortical cells, suggesting a physiological role in adrenocyte migration. Their distribution in the adrenal gland is quite distinct. LN is uniformly present in the steroidogenic cells from the three zones, whereas FN is abundant in the fibrovascular structures of the capsule and the cortex. ACTH treatment of adrenocortical cells strongly induces the expression and secretion of thrombospondin-2 (TSP2), a large trimeric matricellular protein. The multimodular structure of TSP2 is the support of a variety of biological functions. TSP2 promotes attachment but prevents spreading of adrenocortical cells. On the other hand, TSP2 induces the activation of latent TGFbeta through an indirect mechanism and is anti-angiogenic in vitro. The overall distribution of TSP2 in the glomerulosa and fasciculata zones of the adrenal cortex, and its absence from the reticularis zone, argue in favor of a role in the protection of adrenocortical cells against apoptosis. In the adrenal cortex, five main biological functions are potentially regulated by components of the extracellular matrix : stem cell commitment into the adrenocyte differentiation pathway, terminal differentiation toward the three distinct adrenocyte phenotypes, centripetal migration, apoptosis and the formation of the capillary network. Future studies will aim at deciphering which extracellular component(s) is involved in each of these regulations.
Corticotropin-induced secreted protein (CISP) is a trimeric glycoprotein secreted by primary cultures of bovine adrenortical cells in response to adrenocorticotropic hormone (ACTH). This protein was recently purified in our laboratory, and its N-terminal amino-acid sequence revealed a significant similarity with thrombospondin-2 (TSP2). We report here the nucleotide sequence of a 386 bp RT-PCR fragment specific for CISP. The deduced protein sequence shares 84% identity with the N-terminal portion of mature human TSP2, suggesting that CISP is its bovine counterpart. Northern analysis of adrenocortical cell RNA using the above cDNA fragment as a probe revealed a 6.0 kb CISP/TSP2 mRNA whose abundance was increased nearly fivefold following a 24 h cell treatment with 10(-7) M ACTH. Under the same conditions, the expression of TSP1 mRNA was reduced by tenfold. The protein levels of TSP1 and CISP/TSP2 varied accordingly with their respective mRNA levels, as shown by immunoprecipitation and immunofluorescence experiments. Taken together, these data show that ACTH induces a dramatic shift in the pattern of adrenocortical cell thrombospondin expression from TSP1 to CISP/TSP2. This observation suggests that these two members of the thrombospondin family exert distinct biological functions in the adrenal cortex. This hypothesis is further supported by the observation that anti-CISP antibodies inhibit the maintenance of the morphological changes of bovine adrenocortical cells induced by ACTH, whereas anti-TSP1 antibodies do not.
ACTH is the major trophic factor regulating and maintaining adrenocortical function, affecting such diverse processes as steroidogenesis, cell proliferation, cell migration, and cell survival. We used differential display RT-PCR to identify genes that are rapidly induced by ACTH in the bovine adrenal cortex. Of 42 PCR products differentially amplified from primary cultures of bovine adrenocortical cells treated with 10 nM ACTH, six identified mRNAs that were confirmed by Northern blot analysis to be induced by ACTH. Four of these amplicons encoded noninformative repetitive sequences. Of the other two sequenced amplicons, one encoded a partial sequence for mitochondrial manganese-dependent superoxide dismutase (SOD2), an enzyme that is likely to protect adrenocortical cells from the cytotoxic effects of radical oxygen species generated during steroid biosynthesis. The second was identified as TIS11b (phorbol-12-myristate-13-acetate-inducible sequence 11b)/ERF-1/cMG, a member of the CCCH double-zinc finger protein family. SOD2 induction by ACTH was independent of extracellular steroid concentration or oxidative stress. SOD2 and TIS11b mRNA expressions were rapidly induced by ACTH, reaching a maximal level after 8 h and 3 h of treatment, respectively. These ACTH effects were mimicked by forskolin but appeared independent of cortisol secretion. Upon ACTH treatment, induction of TIS11b expression closely followed the previously characterized peak of vascular endothelial growth factor (VEGF) expression. Transfection of a TIS11b expression plasmid into 3T3 fibroblasts induced a decrease in the expression of a reporter gene placed upstream of the VEGF 3'-untranslated region, indicating that TIS11b may be an important regulator of VEGF expression through interaction with its 3'-untranslated region.
The adult mammalian adrenal cortex undergoes permanent regeneration. This process implies a cellular proliferation step restricted to the external zone of the tissue, and a subsequent centripetal cell migration during which phenotypic transition from glomerulosa into fasciculata and reticularis cells and elimination of senescent cells through apoptosis occur. As the molecular mechanisms implied in adrenocortical cell migration are still generally unknown, we addressed that question in the present study. Of several extracellular matrix proteins tested, laminin was the most potent chemotactic and haptotactic factor for bovine fasciculata adrenocortical cells. The maximal chemotactic effect (3-fold stimulation) was observed with 50-75 micrograms/ml laminin, whereas the haptotactic effect (3.5-fold stimulation) plateaued for laminin concentrations in the coating solution over 25 micrograms/ml. Using an anti-Engelbreth-Holm-Swarm laminin antibody, we could demonstrate that adrenocortical cells actively synthesize and secrete Engelbreth-Holm-Swarm-laminin, with the A chain produced in limiting quantities. ACTH treatment of adrenocortical cells specifically induced a 2.7- to 4.5-fold increase in A chain synthesis, resulting in a corresponding increase in the amount of secreted laminin. The distribution of laminin in the adrenal cortex tissue was then evaluated by standard immunohistochemistry. The protein appeared to be uniformly expressed in the three zones of the cortex. This observation does not favor the hypothesis that laminin acts as an attractant driving centripetal cell migration. Laminin, which is synthesized under the control of the systemic hormone ACTH, appears as a permissive factor that facilitates proper homeostasis of the adrenocortical tissue.
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