Silvana Balzan

Itraconazole inhibits HMEC-1 angiogenesis.

Abnormal angiogenesis is implicated in a number of human diseases and endothelial growth inhibition represents a common approach in tumor therapy. Recently itraconazole, frequently used in humans as antifungal drug, which blocks the biosynthesis of cholesterol, has been found to be antiangiogenic in primary umbilical vein endothelial cells. However, the exact antiangiogenic mechanisms remain largely unknown. In this paper, we studied the effect of itraconazole in human dermal microvascular endothelial cells (HMEC-1), an immortalized cell line to study adult angiogenesis. A 50% reduction of microtubule formation was observed after itraconazole treatment which was partially rescued by cholesterol addition. We found that itraconazole inhibits angiogenesis markers such as VEGF, AAMP and e-NOS. mTOR and ERK1/2 phosphorylation as well as the expression of Gli1, one of the main controllers of the Shh pathway, were also inhibited by itraconazole. Cholesterol addition did not completely rescue inhibition of these pathways, suggesting that the itraconazole antiangiogenic activity could be due to multiple mechanisms. Our results may contribute to novel approaches to block angiogenesis with therapeutic application.

LH, Progesterone, and TSH can Stimulate Aldosterone In Vitro: A Study on Normal Adrenal Cortex and Aldosterone Producing Adenoma

Endocrine factors different from ACTH or angiotensin II can stimulate aldosterone secretion and have a role in the pathophysiology of hyperaldosteronism. Aldosterone may increase in luteotropic/progestogenic and in hypothyroid states; LH and, occasionally, TSH receptors have been detected in normal adrenal cortex and aldosterone-producing adenoma. The aim of the study was to compare adrenal contents of LH and TSH receptors between normal cortex and aldosterone-producing adenoma and to evaluate the ability of LH, its product progesterone, and TSH to stimulate aldosterone secretion in vitro from primary adrenocortical cells. Surgical aldosterone-producing adenoma fragments from 19 patients and adrenal cortex fragments from 10 kidney donors were used for Western blotting and cell cultures. LH (n=26), TSH (n=19) and progesterone (n=8) receptor proteins were investigated; LH receptor-mRNA was also tested in 8 samples. Aldosterone responses in vitro to LH, progesterone, and TSH stimulation were assayed. LH and TSH receptors were more expressed in adenoma than normal cortex (p<0.01, p<0.05, respectively); progesterone receptor was observed in 6/8 samples. Aldosterone increased after in vitro stimulation with LH (5/12 adenoma, 1/7 normal cells), progesterone (4/5 adenoma, 5/6 normal cells), and TSH (3/5 adenoma and 3/5 normal cells). LH and TSH receptors were more expressed in aldosterone producing adenoma than normal adrenal cortex. LH, progesterone, and TSH can stimulate aldosterone in vitro. Similar mechanisms could participate in vivo in the aldosterone increase in lutheotropic, progestogenic, or hypothyroid states and may exist in both normal adrenal cortex and adenoma in responsive individuals.

Thyroid hormone deiodinases D1, D2, and D3 are expressed in human endothelial dermal microvascular line: effects of thyroid hormones

Endothelial system acts as a large endocrine organ in the human body; however, little is still known about the regulative role of THs on endothelial cells. Aim of the present study was to investigate the expression of the TH deiodinases (D1, D2, and D3) and TH receptors (TRα1, TRα2, and TRβ1) in an endothelial microvascular cultured cell model (HMEC-1), after stimulation with triiodothyronine (T3, 10–100xa0nM), thyroxine (T4, 10–100xa0nM), and reverse T3 (rT3, 1–10xa0nM). DIO1 was significantly inhibited by T4 at 10 and 100xa0nM (pxa0<xa00.001). rT3 significantly inhibited DIO1 at 1xa0nM concentration (pxa0<xa00.01) and stimulated DIO1 at 10xa0nM dosage (pxa0<xa00.001). T4 and rT3 significantly inhibited DIO2 at all concentrations. DIO3 was induced at 100xa0nM T3 (pxa0<xa00.05) and 100xa0nM rT3 (pxa0<xa00.01), and TRα1 and TRα2 mRNAs were significantly increased after 100xa0nM T3 treatment (pxa0<xa00.05) and decreased after 1 and 10xa0nM rT3 (pxa0<xa00.05). TRβ1 was significantly increased by all THs at different concentrations: 10xa0nM T3 and 100xa0nM T3 (pxa0<xa00.05), 1xa0nM rT3 (pxa0<xa00.001), and 100xa0nM T4 (pxa0<xa00.01). D1 and D2 protein levels were evaluated, but no significant difference was observed for any hormonal treatment. For the first time, we found that the TH deiodinases and receptors are expressed in endothelial HMEC-1 cells. These findings might be of significant clinical relevance, given the important regulatory role of the endothelium as first barrier to the bloodstream.

The Stimulative Effect of T3 and T4 on Human Myocardial Endothelial Cell Proliferation, Migration and Angiogenesis

Angiogenesis, the process involving the growth of new blood vessels from pre-existing vessels, may be a target for combating diseases characterized by either poor vascularisation or abnormal vasculature as in cardiovascular diseases. Thyroid hormones (THs) are strong proangiogenic factors whose action starts at the plasma membrane integrin αvβ3 protein transducing rapid nongenomic signals on tumor thyroid cells. The tetraiodothyroacetic acid (Tetrac) inhibits the binding of TH to integrin receptor αVs3 blocking angiogenesis. Growing evidences suggest that, also in heart, Triiodothyronine (T3) and Thyroxine (T4) triggered nongenomic pathways through their binding to integrin receptor αVβ3 inducing capillary proliferation. The angiogenic activity of T3 and T4 has been studied by the chick choriallontoic endothelial cell microtubule assay and the human dermal microvascular endothelial cells microtubule assay. nThe aim of this work was to evaluate the direct stimulative activity of T3 and T4 on human cardiac microvascular endothelial cell (HMVEC-C) proliferation, migration and tube formation, employing Tetrac as inhibitor. Our in vitro study indicates that T3 and T4 directly stimulate angiogenesis in HMVEC-C observed as capillary density, cell proliferation and cell migration. In all models, Tetrac (5 μM) inhibited the proangiogenic effect of T3 and T4 suggesting its integrin-mediated action. Sq RT-PCR assay revealed that T3, and in less extent T4, increased the expression of angiogenic genes such as angiopoietin-1 (Angpt-1), angio-associated migratory cell protein (AAMP) and vascular endothelial growth factor (VEGF), whereas when the cells were pre-incubated with Tetrac this effect was abolished. In conclusion, our results show that THs stimulate angiogenesis, suggesting a potential therapeutic role aimed at increasing capillary density in cardiac diseases.

Modification of gene expression profiling related to renin–angiotensin system in an ischemia/reperfusion rat model after T3 infusion

Triiodothyronine (T3) and renin–angiotensin system (RAS) are functionally related in cardiovascular system. Recently, in an in vivo myocardial ischemia/reperfusion (I/R) model in rats, we showed that T3 treatment improved the post-ischemic recovery of cardiac function. In the present study, we used the same experimental model of regional I/R, obtained by 30xa0min occlusion of the left descending coronary artery, followed by 3-days of reperfusion, to investigate the effect of 48-h treatment (started 1xa0day after ischemia) with 6xa0µg/kg/day T3 or vehicle. T3 was delivered by constant subcutaneous infusion via miniosmotic pump. In particular, aim of this work is to evaluate the effects of T3 on the gene expression of the main receptors and enzymes involved in the two cardiac arms of RAS in an in vivo rat model of I/R: AT1R-ACE (detrimental arm) and AT2R/MAS1-ACE2 (protective arm). Gene expression was evaluated by Real-Time PCR in infarct zone (Area-At-Risk: AAR) and in tissues distant from ischemic wound (Remote Zone: RZ). Three different rat groups were used: sham-operated; I/R and I/Ru2009+u2009T3. Main result of the study is the opposite response of AT1R and AT2R/MAS1 expression to I/R procedure and to T3 administration after I/R in both AAR and RZ. Moreover, T3 significantly increased ACE and ACE2 enzyme expression in AAR and RZ. This study reveals that T3 stimulates the expression of protective genes related to RAS such as AT2R/MAS1-ACE2 mainly in BZ, suggesting that, at least in part, T3 could be involved in the local cardiac ameliorative response to I/R procedure.

[PP.27.20] EVIDENCE FOR PERSISTENT ORGANOCHLORINE POLLUTANTS IN HUMAN NORMAL ADRENAL CORTEX AND ALDOSTERONE-PRODUCING ADENOMA

Objective: Various stimuli may influence adrenal cortex hormonogenesis and have a role in the pathophysiology of human endocrine dysfunction. Environmental pollutants may act as endocrine disruptors. Organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) may enter the food chain and accumulate in animal tissues including adrenals, where they can exert endocrine dysregulating effects and promote cell proliferation as observed in animal and in cell culture studies (Harvey, 2016). To our knowledge, no previous study investigated their presence in the human adrenal cortex. Our aim was to investigate, in vitro, the presence in the human adrenal cortex of selected OCPs and PCBs, in a comparison between normal adrenals (NA) and aldosterone-producing adenomas (APA). Design and method: After informed consent, surgical fragments of APA from 11 hypertensive patients (6F/5u200aM, age 61u200a±u200a10 yrs) and NA from 8 kidney donors (5F/3u200aM, age 54u200a±u200a14 yrs) were assayed for sixteen PCBs congeners (n 28, 31, 52, 66, 77, 101, 105, 118, 126, 128, 138, 153, 156 169, 170 and 180) and eight OCPs (alpha, beta, gamma exachlorocycloexane, exachlorobenzene and the DDTderivatives ppDDT, opDDT, ppDDD, ppDDE). A Matrix solid-phase dispersion method coupled with gas-chromatography triple quadrupole mass spectrometry were used for their simultaneous determination (Turci, 2012). A concentration equal to 0.05u200a&mgr;g/g was arbitrarily assigned to samples showing concentrations at the limit of the detection accuracy. Data were analyzed by means of Random Forest and Wilcoxon rank-sum test. Results: OCPs and PCBs were found in both adrenocortical cell types. A subset of pollutants was found to be more characterizing APA than NA. Higher concentrations (&mgr;g/g) in APA were observed for alpha, beta, gamma exachlorocycloexane (1.48u200a±u200a3.32 vs 0.17u200a±u200a0.19, pu200a=u200a0.028; 2.81u200a±u200a2.10 vs 0.96u200a±u200a0.98, pu200a=u200a0.011; 2.16u200a±u200a4.85 vs 0.17u200a±u200a0.26, pu200a=u200a0.004, respectively), as well as for exachlorobenzene and PCBs 28, 52, and 101 (3.41u200a±u200a3.11 vs 0.97u200a±u200a1.06, pu200a=u200a0.021; 2.34u200a±u200a4.68 vs 0.25u200a±u200a0.22, pu200a=u200a0.039; 0.58u200a±u200a1.19 vs 0.06u200a±u200a0.02, pu200a=u200a0.002; 0.26u200a±u200a0.43 vs 0.05u200a±u200a0.00, pu200a=u200a0.001, respectively). Conclusions: To our knowledge, this is the first study showing the presence of organochlorine pollutants in human adrenal cortex. Dedicated studies are deserved to characterize their biological relevance and investigate their possible causative role in human adrenal disease.

Evidence for persistent organochlorine pollutants in the human adrenal cortex

Environmental pollutants may act as endocrine disruptors in animals. Organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) enter the food chain and may accumulate in the fatty animal tissues, including adrenals. To our knowledge, no previous study has investigated their presence in the human normal adrenal (NA) cortex and aldosterone‐producing adenomas (APA). Surgical fragments of APA from 11 patients and NA from 8 kidney donors were analyzed for 16 PCBs congeners and 10 OCPs. A Matrix Solid‐Phase Dispersion (MSPD) method for simultaneous determination of the target compounds in cortex homogenates was developed. A gas‐chromatography triple quadrupole mass spectrometry (Triple Quad GC‐MS) system was used for the analysis. Data were analyzed using Random Forest and Wilcoxons rank‐sum test. OCPs and PCBs were found in specimens from both types. A subset of pollutants characterized APA more than NA. Higher concentrations (μg g−1) in APA were observed for α‐, β‐, and γ‐ Hexachlorocyclohexane (HCH) (1.48 ± 3.32 vs. 0.17 ± 0.19, P = 0.028; 2.81 ± 2.10 vs. 0.96 ± 0.98, P = 0.011; 2.16 ± 4.85 vs. 0.17 ± 0.26, P = 0.004, respectively), as well as for Hexachlorobenzene (HCB) and for PCBs 28, 52 and 101 (3.41 ± 3.11 vs. 0.97 ± 1.06, P = 0.021; 2.34 ± 4.68 vs. 0.25 ± 0.22, P = 0.039; 0.58 ± 1.19 vs. 0.06 ± 0.02, P = 0.002; 0.26 ± 0.43 vs. 0.05 ± 0.00, P = 0.001, respectively). Environmental organochlorine pollutants were shown to be present in the human normal and abnormal adrenal cortex, deserving future investigation on their possible role as adrenal endocrine disruptors in human disease. Copyright