Role of androgens in vascular smooth muscle cell calcification

University of Technology, Sydney. Faculty of Science.Calcification is a common feature of advanced atherosclerotic lesions and is a clinically significant predictor of cardiovascular events. Coronary calcification is more prevalent in men than age-matched women. However, atherosclerotic calcification increases in postmenopausal women, who present with lower levels of estrogen, suggesting that sex hormones play a critical role in its pathogenesis and progression. This has implications for hormone therapy treatment that is used to treat age-related conditions such as osteoporosis and menopause Extensive observational studies into estrogen replacement therapy have revealed that postmenopausal women treated with estrogen exhibit less extensive atherosclerotic calcification. The effects of androgens on atherosclerotic calcification have, however, received little attention and consequently its mechanisms remain poorly understood. This study therefore explored the effects of androgens on atherosclerotic calcification. In vitro studies postulate vascular smooth muscle cell (VSMC) differentiation into mineralising osteoblast-like cells as a key mediator of atherosclerotic calcification. Given the gen der disparity in atherosclerotic calcification we hypothesised that androgens promote differentiation of VSMC into mineralising osteoblast-like cells. Th erefore, the aims of this st udy wer e to 1) examine the effects of androgens in vascular smooth muscle cell differentiation and calcification and 2) elucidate the molecular mechanisms of androgen action in this process, using phosphate-induced bovine and murine in vitro models of calcification. This study demonstrated that co-treatment of bovine coronary artery smooth muscle cells (BCASMC) with phosphate and testosterone (T) and dihydrotestosterone (DHT) promoted calcification. Investigation of the molecular mechanisms underlying calcification in the bovine model revealed Tstimulated calcification was estrogen receptor (ER) driven. DHT, however, mediated its effects via the androgen receptor (AR). Further investigation of molecular mechanisms showed DHT regulated ALP activity whereas T did not. T, therefore, promoted calcification in an ER-driven, ALP independent pathway in contrast to DHT, which mediated its effects via an AR-driven, ALP dependent pathway. A primary mouse cell-based calcification model was also established. In contrast to the bovine model, it was found that T and DHT treatment did not promote calcification in the murine model. The lack of androgen promotion of calcification in this model was associated with the absence of ALP activity. The conclusion drawn from the bovine model, of a mechanistic role for ALP in the DHT /AR driven mineralisation but not for T-driven mineralisation, suggested that in the murine cells an ER pathway is not functioning. In conclusion, the studies presented in this thesis demonstrate that T and DHT promote differentiation of vascular smooth muscle cells into osteoblast-like cells capable of mineralisation. T and DHT mediate calcification via alternative pathways that can involve AR and ERs. A potential mechanistic role for ALP in DHT /AR-driven mineralisation has been established

Reconstituted high-density lipoproteins promote wound repair and blood flow recovery in response to ischemia in aged mice

Background: The average population age is increasing and the incidence of age-related vascular complications is rising in parallel. Impaired wound healing and disordered ischemia-mediated angiogenesis are key contributors to age-impaired vascular complications that can lead to amputation. High-density lipoproteins (HDL) have vasculo-protective properties and augment ischemia-driven angiogenesis in young animals. We aimed to determine the effect of reconstituted HDL (rHDL) on aged mice in a murine wound healing model and the hindlimb ischemia (HLI) model. Methods: Murine wound healing model—24-month-old aged mice received topical application of rHDL (50 μg/wound/ day) or PBS (vehicle control) for 10 days following wounding. Murine HLI model—Femoral artery ligation was performed on 24-month-old mice. Mice received rHDL (40 mg/kg) or PBS, intravenously, on alternate days, 1 week pre-surgery and up to 21 days post ligation. For both models, blood flow perfusion was determined using laser Doppler perfusion imaging. Mice were sacrificed at 10 (wound healing) or 21 (HLI) days post-surgery and tissues were collected for histological and gene analyses. Results: Daily topical application of rHDL increased the rate of wound closure by Day 7 post-wounding (25 %, p < 0.05). Wound blood perfusion, a marker of angiogenesis, was elevated in rHDL treated wounds (Days 4–10 by 22–25 %, p < 0. 05). In addition, rHDL increased wound capillary density by 52.6 %. In the HLI model, rHDL infusions augmented blood flow recovery in ischemic limbs (Day 18 by 50 % and Day 21 by 88 %, p < 0.05) and prevented tissue necrosis and toe loss. Assessment of capillary density in ischemic hindlimb sections found a 90 % increase in rHDL infused animals. In vitro studies in fibroblasts isolated from aged mice found that incubation with rHDL was able to significantly increase the key pro-angiogenic mediator vascular endothelial growth factor (VEGF) protein (25 %, p < 0.05). Conclusion: rHDL can promote wound healing and wound angiogenesis, and blood flow recovery in response to ischemia in aged mice. Mechanistically, this is likely to be via an increase in VEGF. This highlights a potential role for HDL in the therapeutic modulation of age-impaired vascular complications

The regulation of miRNAs by reconstituted high-density lipoproteins in diabetes-impaired angiogenesis

Diabetic vascular complications are associated with impaired ischaemia-driven angiogenesis. We recently found that reconstituted high-density lipoproteins (rHDL) rescue diabetes-impaired angiogenesis. microRNAs (miRNAs) regulate angiogenesis and are transported within HDL to sites of injury/repair. The role of miRNAs in the rescue of diabetes-impaired angiogenesis by rHDL is unknown. Using a miRNA array, we found that rHDL inhibits hsa-miR-181c-5p expression in vitro and using a hsa-miR-181c-5p mimic and antimiR identify a novel anti-angiogenic role for miR-181c-5p. miRNA expression was tracked over time post-hindlimb ischaemic induction in diabetic mice. Early post-ischaemia when angiogenesis is important, rHDL suppressed hindlimb mmu-miR-181c-5p. mmu-miR-181c-5p was not detected in the plasma or within HDL, suggesting rHDL specifically targets mmu-miR-181c-5p at the ischaemic site. Three known angiogenic miRNAs (mmu-miR-223-3p, mmu-miR-27b-3p, mmu-miR-92a-3p) were elevated in the HDL fraction of diabetic rHDL-infused mice early post-ischaemia. This was accompanied by a decrease in plasma levels. Only mmu-miR-223-3p levels were elevated in the hindlimb 3 days post-ischaemia, indicating that rHDL regulates mmu-miR-223-3p in a time-dependent and site-specific manner. The early regulation of miRNAs, particularly miR-181c-5p, may underpin the rescue of diabetes-impaired angiogenesis by rHDL and has implications for the treatment of diabetes-related vascular complications

Estrogen Receptor Control of Atherosclerotic Calcification and Smooth Muscle Cell Osteogenic Differentiation

© 2017 American Heart Association, Inc. Objective-Vascular calcification is associated with increased risk of myocardial infarction and stroke. The objective of this work was to examine the ability of 17β-estradiol (E2) to stimulate calcification of vascular smooth muscle cells (VSMC) in vivo, using aged apolipoprotein E-null mice with advanced atherosclerotic lesions, and subsequently to explore underlying mechanisms in vitro. Approach and Results-Silastic E2 capsules were implanted into male and female apolipoprotein E-null mice aged 34 weeks. Plaque and calcified area were measured in the aortic sinus and innominate artery after 8 weeks. Immunohistochemical analysis examined expression of the estrogen receptors (estrogen receptor alpha and estrogen receptor beta [ERβ]). VSMC expression of osteogenic markers was examined using digital polymerase chain reaction. Advanced atherosclerotic lesions were present in all mice at the end of 8 weeks. In both male and female mice, E2 increased calcified area in a site-specific manner in the aortic sinus independently of plaque growth or lipid levels and occurred in association with a site-specific decrease in the proportion of ERβ-positive intimal cells. Calcified lesions expressed collagen I and bone sialoprotein, with decreased matrix Gla protein. In vitro, E2 suppressed ERβ expression and increased VSMC mineralization, demonstrating increased collagen I and II, osteocalcin and bone sialoprotein, and reduced matrix Gla protein and osteopontin. Antagonism or RNA silencing of estrogen receptor alpha, ERβ, or both further increased VSMC mineralization. Conclusions-We have demonstrated that E2 can drive calcification in advanced atherosclerotic lesions by promoting the differentiation of VSMC to osteoblast-like cells, a process which is augmented by inhibition of estrogen receptor alpha or ERβ activity

Transcription-targeted gene therapy for androgen-independent prostate cancer

The Escherichia coli enzyme (purine nucleoside phosphorylase, PNP) gene is delivered directly into PC3 tumors by one injection of replication-deficient human type-5 adenovirus (Ad5). Expressed PNP converts the systemically administered prodrug, 6MPDR, to a toxic purine, 6MP, causing cell death. We sought to increase the specificity of recombinant Ad vectors by controlling PNP expression with the promoter region from the androgen-dependent, prostate-specific rat probasin (Pb) gene. To increase its activity, the promoter was combined with the SV40 enhancer (SVPb). Cell lines were transfected with plasmids containing both a reporter gene, under SVPb control, and a reference gene cassette to allow normalization of expression levels. Plasmids expressed ∼20-fold more reporter in prostate cancer than in other cells, but surprisingly, the SVPb element was both androgen-independent and retained substantial prostate specificity. Killing by Ad5-SVPb-PNP vector of cell lines cultured with 6MPDR for 6 days was 5- to 10-fold greater in prostate cancer than in liver or lung cells. In vivo, a single intratumoral injection of Ad5-SVPb-PNP (4×108 pfu), followed by 6MPDR administration twice daily for 6 days, significantly suppressed the growth of human prostate tumors in nude mice and increased their survival compared to control animals. Thus, the androgen-independent, prostate-targeting Ad5 vector reduces human prostate cancer growth significantly in vitro and in vivo. This first example of an androgen-independent vector points the way toward treatment of emerging androgen-independent prostate cancer in conjunction with hormone ablation therapy at a time when the tumor burden is low

Strikingly different atheroprotective effects of apolipoprotein A-I in early- versus late-stage atherosclerosis

Preclinical studies have shown benefit of apolipoprotein A-I (apoA-I)/high-density lipoprotein (HDL) raising in atherosclerosis; however, this has not yet translated into a successful clinical therapy. Our studies demonstrate that apoA-I raising is more effective at reducing early-stage atherosclerosis than late-stage disease, indicating that the timing of HDL raising is a critical factor in its atheroprotective effects. To date, HDL-raising clinical trials have only been performed in aged patients with advanced atherosclerotic disease. Our findings therefore provide insight, related to important temporal aspects of HDL raising, as to why the clinical trials have thus far been largely neutral.Jamie Morton, Shisan Bao, Laura Z. Vanags, Tania Tsatralis, Anisyah Ridiandries, Chung-Wah Siu, Kwong-Man Ng, Joanne T.M. Tan, David S. Celermajer, Martin K.C. Ng, Christina A. Bursil

The regulation of miRNAs by reconstituted high-density lipoproteins in diabetes-impaired angiogenesis

Diabetic vascular complications are associated with impaired ischaemia-driven angiogenesis. We recently found that reconstituted high-density lipoproteins (rHDL) rescue diabetes-impaired angiogenesis. microRNAs (miRNAs) regulate angiogenesis and are transported within HDL to sites of injury/repair. The role of miRNAs in the rescue of diabetes-impaired angiogenesis by rHDL is unknown. Using a miRNA array, we found that rHDL inhibits hsa-miR-181c-5p expression in vitro and using a hsa-miR-181c-5p mimic and antimiR identify a novel anti-angiogenic role for miR-181c-5p. miRNA expression was tracked over time post-hindlimb ischaemic induction in diabetic mice. Early post-ischaemia when angiogenesis is important, rHDL suppressed hindlimb mmu-miR-181c-5p. mmu-miR-181c-5p was not detected in the plasma or within HDL, suggesting rHDL specifically targets mmu-miR-181c-5p at the ischaemic site. Three known angiogenic miRNAs (mmu-miR-223-3p, mmu-miR-27b-3p, mmu-miR-92a-3p) were elevated in the HDL fraction of diabetic rHDL-infused mice early post-ischaemia. This was accompanied by a decrease in plasma levels. Only mmu-miR-223-3p levels were elevated in the hindlimb 3 days post-ischaemia, indicating that rHDL regulates mmu-miR-223-3p in a time-dependent and site-specific manner. The early regulation of miRNAs, particularly miR-181c-5p, may underpin the rescue of diabetes-impaired angiogenesis by rHDL and has implications for the treatment of diabetes-related vascular complications.Samuel T. Hourigan, Emma L. Solly, Victoria A. Nankivell, Anisyah Ridiandries, Benjamin M. Weimann, Rodney Henriquez, Edward R. Tepper, Jennifer Q. J. Zhang, Tania Tsatralis, Zoe E. Clayton, Laura Z. Vanags, Stacy Robertson, Stephen J. Nicholls, Martin K. C. Ng, Christina A. Bursill, Joanne T. M. Ta

High-density lipoproteins rescue diabetes-impaired angiogenesis via scavenger receptor Class B Type I

Disordered neovascularization and impaired wound healing are important contributors to diabetic vascular complications. We recently showed that high-density lipoproteins (HDLs) enhance ischemia-mediated neovascularization, and mounting evidence suggests HDL have antidiabetic properties. We therefore hypothesized that HDL rescue diabetes-impaired neovascularization. Streptozotocin-induced diabetic mice had reduced blood flow recovery and neovessel formation in a hindlimb ischemia model compared with nondiabetic mice. Reconstituted HDL (rHDL) infusions in diabetic mice restored blood flow recovery and capillary density to nondiabetic levels. Topical rHDL application rescued diabetes-impaired wound closure, wound angiogenesis, and capillary density. In vitro, rHDL increased key mediators involved in hypoxia-inducible factor-1α (HIF-1α) stabilization, including the phosphoinositide 3-kinase/Akt pathway, Siah1, and Siah2, and suppressed the prolyl hydroxylases (PHD) 2 and PHD3. rHDL rescued high glucose–induced impairment of tubulogenesis and vascular endothelial growth factor (VEGF) A protein production, a finding associated with enhanced phosphorylation of proangiogenic mediators VEGF receptor 2 and endothelial nitric oxide synthase. Siah1/2 small interfering RNA knockdown confirmed the importance of HIF-1α stability in mediating rHDL action. Lentiviral short hairpin RNA knockdown of scavenger receptor class B type I (SR-BI) in vitro and SR-BI−/− diabetic mice in vivo attenuated rHDL rescue of diabetes-impaired angiogenesis, indicating a key role for SR-BI. These findings provide a greater understanding of the vascular biological effects of HDL, with potential therapeutic implications for diabetic vascular complications.Joanne T.M. Tan, Hamish C.G. Prosser, Louise L. Dunn, Laura Z. Vanags, Anisyah Ridiandries, Tania Tsatralis, Laura Lecce, Zoë E. Clayton, Sui Ching G. Yuen, Stacy Robertson, Yuen Ting Lam, David S. Celermajer, Martin K.C. Ng and Christina A. Bursil

VEGFR2 is activated by high-density lipoproteins and plays a key role in the proangiogenic action of HDL in ischemia

High-density lipoproteins augment hypoxia-induced angiogenesis by inducing the key angiogenic vascular endothelial growth factor A (VEGFA) and total protein levels of its receptor 2 (VEGFR2). The activation/phosphorylation of VEGFR2 is critical for mediating downstream, angiogenic signaling events. This study aimed to determine whether reconstituted high-density lipoprotein (rHDL) activates VEGFR2 phosphorylation and the downstream signaling events and the importance of VEGFR2 in the proangiogenic effects of rHDL in hypoxia. In vitro, rHDL increased VEGFR2 activation and enhanced phosphorylation of downstream, angiogenic signaling proteins ERK1/2 and p38 MAPK in hypoxia. Incubation with a VEGFR2-neutralizing antibody attenuated rHDL-induced phosphorylation of VEGFR2, ERK1/2, p38 MAPK, and tubule formation. In a murine model of ischemia-driven neovascularization, rHDL infusions enhanced blood perfusion and augmented capillary and arteriolar density. Infusion of a VEGFR2-neutralizing antibody ablated those proangiogenic effects of rHDL. Circulating Sca1+/CXCR4+ angiogenic progenitor cell levels, important for neovascularization in response to ischemia, were higher in rHDL-infused mice 3 d after ischemic induction, but that did not occur in mice that also received the VEGFR2-neutralizing antibody. In summary, VEGFR2 has a key role in the proangiogenic effects of rHDL in hypoxia/ischemia. These findings have therapeutic implications for angiogenic diseases associated with an impaired response to tissue ischemia.Carla M. Cannizzo, Aaron A. Adonopulos, Emma L. Solly, Anisyah Ridiandries, Laura Z. Vanags, Jocelyne Mulangala, Sui Ching G. Yuen, Tania Tsatralis, Rodney Henriquez, Stacy Robertson, Stephen J. Nicholls, Belinda A. Di Bartolo, Martin K.C. Ng, Yuen Ting Lam, Christina A. Bursill, and Joanne T.M. Ta