Vascular interventions result in the disruption of the tunica intima and the exposure of sub-endothelial matrix proteins. Nanoparticles designed to bind to these exposed matrices could provide targeted drug delivery systems aimed at inhibiting dysfunctional vascular remodeling and improving intervention outcomes. Here, we present the progress in the development of targeted liposomal nanocarriers designed for preferential collagen IV binding under simulated static vascular flow conditions. PEGylated liposomes (PLPs), previously established as effective delivery systems in vascular cells types, served as non-targeting controls. Collagen-targeting liposomes (CT-PLPs) were formed by conjugating established collagen-binding peptides to modified lipid heads via click chemistry (CTL), and inserting them at varying mol% either at the time of PLP assembly or via micellar transfer. All groups included fluorescently labeled lipid species for imaging and quantification. Liposomes were exposed to collagen IV matrices statically or via hemodynamic flow, and binding was measured via fluorometric analyses. CT-PLPs formed with 5 mol% CTL at the time of assembly demonstrated the highest binding affinity to collagen IV under static conditions, while maintaining a nanoparticle characterization profile of ~50 nm size and a homogeneity polydispersity index (PDI) of ~0.2 favorable for clinical translation. When liposomes were exposed to collagen matrices within a pressurized flow system, empirically defined CT-PLPs demonstrated significant binding at shear stresses mimetic of physiological through pathological conditions in both the venous and arterial architectures. Furthermore, when human saphenous vein explants were perfused with liposomes within a closed bioreactor system, CT-PLPs demonstrated significant ex vivo binding to diseased vascular tissue. Ongoing studies aim to further develop CT-PLPs for controlled targeting in a rodent model of vascular injury. The CT-PLP nanocarriers established here show promise as the framework for a spatially controlled delivery platform for future application in targeted vascular therapeutics.

Lipid nanoparticles have become increasingly popular delivery platforms in the field of gene therapy, but bench-to-bedside success has been limited. Many liposomal gene vectors are comprised of synthetic cationic lipids, which are associated with lipid-induced cytotoxicity and immunogenicity. Natural, non-cationic PEGylated liposomes (PLPs) demonstrate favorable biocompatibility profiles but are not considered viable gene delivery vehicles due to inefficient nucleic acid loading and reduced cellular uptake. PLPs can be modified with cell-penetrating peptides (CPPs) to enhance the intracellular delivery of liposomal cargo but encapsulate leakage upon CPP-PLP assembly is problematic. Here, we aimed to identify parameters that overcome these performance barriers by incorporating nucleic acid condensers during CPP-PLP assembly and screening variable ethanol injection parameters for optimization. CPP-PLPs were formed with R8-amphiphiles via pre-insertion, post-insertion and post-conjugation techniques and liposomes were characterized for size, surface charge, homogeneity, siRNA encapsulation efficiency and retention and cell associative properties. Herein we demonstrate that pre-insertion of stearylated R8 into PLPs is an efficient method to produce non-cationic CPP-PLPs and we provide additional assembly parameter specifications for a modified ethanol injection technique that is optimized for siRNA encapsulation/retention and enhanced cell association. This assembly technique could provide improved clinical translation of liposomal based gene therapy applications.

Background: Intimal hyperplasia (IH) is the most common indicator for secondary intervention in peripheral vascular disease. Matrix metalloproteinases (MMPs) play a role in IH development due to their degradation of the extracellular matrix. Doxycycline (Doxy), a member of the tetracycline family of antibiotics, is a potent MMP inhibitor. We have previously shown that Doxy inhibits MMP activity and vascular smooth muscle cell migration in vitro. We hypothesized that Doxy would decrease MMP activity in vivo and inhibit the development of IH in a rodent model of vascular injury. Methods and Results: Doxy (400 mg/pellet) was delivered by a slow-release pellet implanted 3 days prior to or at the time of balloon angioplasty (BA) of the common carotid artery in female rats. At 14 days post-BA, intima-to-media (I:M) ratios were 0.77 +/- 0.21 and 1.04 +/- 0.32 in the Doxy treated groups, respectively, compared to 1.25 +/- 0.26 in the control group (P = not significant; n = 3). Additionally, the tested dose of Doxy in either group had no inhibitory effect on membrane type 1-MMP or MMP-2 tissue levels, as measured by immunohistochemistry, or on systemic levels of MMP, as measured by total MMP serum levels using enzyme-linked immunosorbent assay. At 14 days post-BA, VSMC proliferation in the injured artery was increased to Doxy treatment prior to and at the time of surgery (23.5 +/- 3.4 and 27.2 +/- 3.9%, respectively), compared to control (11.4 +/- 0.4%; n = 3), as measured by proliferating cellular nuclear antigen immunostaining. Conclusions: In our in vivo model of vascular injury, systemic Doxy administration prior to or at the time of vascular injury does not significantly hinder the progression of IH development. Additional doses and routes of administration could be examined in order to correlate therapeutic serum levels of Doxy with effective MMP inhibition in serum and arterial tissue. However, alternative drug delivery systems are needed in order to optimize therapeutic administration of targeted MMP inhibitors for the prevention of IH development.

The polyherbal blend Zyflamend™ has been shown to have anti-inflammatory properties and attenuate inflammatory-modulated pathologies. Fish oils have also been shown to have cardioprotective properties. However, the beneficial effects of their combination have not been investigated. Intimal hyperplasia (IH), a pathological remodeling response of a vessel to injury, is heavily regulated by an immune-mediated reaction. The objective of this study was to determine if dietary supplementation with Zyflamend and/or Wholemega could affect inflammatory-dependent vascular remodeling mechanisms when provided at human equivalent doses. Based on their anti-inflammatory properties and protective benefits demonstrated in previous pre-clinical studies, we hypothesized administration of these supplements would prevent IH in an animal model of vascular injury. The diets of aged male rats were supplemented with human equivalent doses of Zyflamend (Zyf) and/or Wholemega (WMega) or placebo (Plac) for 1wk prior to balloon angioplasty (BA)-induced injury of the left carotid artery. At 28d post-injury morphometric analysis of carotid tissue revealed IH was decreased in Zyf + WMega animals compared to placebo, while Zyf or WMega independently had no significant effect. Serum cytokine screening indicated injury-induced interleukin family isoforms, interferon-γ, and macrophage inflammatory proteins were downregulated by Zyf + WMega. Immunohistochemical staining for monocyte/macrophage phenotypic markers revealed that while overall monocyte/macrophage vessel infiltration was not affected, Zyf + WMega limited the alternative differentiation of M2 macrophages and reduced the presence of myofibroblasts in the injured vessel wall. In summary, dietary supplementation with Zyf + WMega attenuated the acute inflammatory response following vascular injury and inhibited IH development in vivo.

Metformin, one of the most commonly used drugs for the treatment of type II diabetes, was recently found to exert its therapeutic effects, at least in part, by activating the AMP-activated protein kinase (AMPK). However, the site of its action, as well as the mechanism to activate AMPK, remains elusive. Here we report how metformin activates AMPK. In cultured bovine aortic endothelial cells, metformin dose-dependently activated AMPK in parallel with increased detection of reactive nitrogen species (RNS). Further, either depletion of mitochondria or adenoviral overexpression of superoxide dismutases, as well as inhibition of nitric-oxide synthase, abolished the metformin-enhanced phosphorylations and activities of AMPK, implicating that activation of AMPK by metformin might be mediated by the mitochondria-derived RNS. Furthermore, administration of metformin, which increased 3-nitrotyrosine staining in hearts of C57BL6, resulted in parallel activation of AMPK in the aorta and hearts of C57BL6 mice but not in those of endothelial nitric-oxide synthase (eNOS) knockout mice in which metformin had no effect on 3-nitrotyrosine staining. Because the eNOS knockout mice expressed normal levels of AMPK-alpha that was activated by 5-aminoimidazole-4-carboxamide riboside, an AMPK agonist, these data indicate that RNS generated by metformin is required for AMPK activation in vivo. In addition, metformin significantly increased the co-immunoprecipitation of AMPK and its upstream kinase, LKB1, in C57BL6 mice administered to metformin in vivo. Using pharmacological and genetic inhibitors, we found that inhibition of either c-Src or PI3K abolished AMPK that was enhanced by metformin. We conclude that activation of AMPK by metformin might be mediated by mitochondria-derived RNS, and activation of the c-Src/PI3K pathway might generate a metabolite or other molecule inside the cell to promote AMPK activation by the LKB1 complex.

Activation of the AMP-activated Protein Kinase by the Anti-diabetic Drug Metformin in Vivo: ROLE OF MITOCHONDRIAL REACTIVE NITROGEN SPECIESJournal of Biological ChemistryVol. 279Issue 42PreviewMetformin, one of the most commonly used drugs for the treatment of type II diabetes, was recently found to exert its therapeutic effects, at least in part, by activating the AMP-activated protein kinase (AMPK). However, the site of its action, as well as the mechanism to activate AMPK, remains elusive. Here we report how metformin activates AMPK. In cultured bovine aortic endothelial cells, metformin dose-dependently activated AMPK in parallel with increased detection of reactive nitrogen species (RNS). Full-Text PDF Open Access VOLUME 279 (2004) PAGES 43940–43951 This article has been withdrawn by the authors. The Journal raised questions that the AMPK immunoblot in Fig. 3C was reused in Fig. 4A as ACC, lanes 1 and 4 of the AMPK-P immunoblot in Fig. 4A were duplicated, and lanes 3 and 4 of the AMPK-P immunoblot in Fig. 4F were reused in Fig. 5A as ACC-P. Fifteen years after publication, the original data for these figures were not available for evaluation. The authors were able to provide to the Journal data from repeat experiments for Fig. 4A performed at the time of the original work, which they state confirm the results. The authors also provided the journal evidence that the duplications of Figs. 3C, 4F, and 5A occurred due to errors in the figure preparation. The authors offered to publish substitute figures based on the repeated experiments and corrected figures, alternatively, offered to repeat the experiments. However, the Journal declined both offers, a decision with which the authors respectfully disagree. Further, the authors state that the results of this article are confirmed by the results of complementary experiments presented in the article, and the principal conclusion was further confirmed in publications from other laboratories (Quintero, M. et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 5379–5384; Guilherme, L. et al. (2006) Diabetes Care 29, 1083–1089). The article, with confirmatory data supporting the results, can be obtained by contacting the authors. The authors stand by the experimental data and the conclusions of the article.

Introduction: Our laboratory aims to develop biocompatible nanocarriers for molecular therapeutics aimed at vascular pathology. We have previously established a liposome platform that is an effective delivery system for RNAi in vascular smooth muscle cells (VSMC). Tailoring liposome membranes to mimic vascular cell membrane lipid constituents may be a promising strategy for increased delivery to target cells. Here we test our previously established liposome platform with the incorporation of naturally occurring signaling lipids known to influence vascular cell function as a method to increase VSMC association. Methods: Established cell-penetrating neutral liposomes (R8-PLPs) were assembled and fluorescently tagged as previously described. The propensity of diacylglycerol (DAG) and/or phosphatidylserine (PS) to increase the association of R8-PLP to VSMCs was tested by the incorporation of gradient percentages DAG/PS alone and in combination at 5-20% membrane occupancy. Liposome stability and siRNA encapsulate retention was analyzed via dynamic light scattering and Ribo-green, respectively. Results: DAG and PS incorporation increased VSMC association of R8-PLP, with 10% PS increased over all other groups (P10; Fig1A). Combinatorial formulations were screened for optimal DAG content with PS fixed at 10%. DAG20%+PS10% (D20P10) performed best, with increased VSMC association over all other combinatorial groups or independent P10 modification (Fig1B). Stability profiles were consistent (~50nm size and ~80% drug retention) and not significantly different among groups. Conclusion: Signaling lipid incorporation into the nanocarrier architecture potentiates VSMC association of established R8-PLP liposomes, without sacrificing stability or drug retention. These results suggest cell mimetic tuning of liposomes to generate specificity and increase delivery efficacy is a viable strategy for advancing targeted liposomal drug delivery.

Vascular intervention results in intimal denudation, exposure of the subendothelial matrix, and subsequent intimal hyperplasia (IH), under the control of numerous remodeling mechanisms. Reduction of IH-induced restenosis may be achieved by manipulation of these remodeling pathways through targeted molecular inhibition. Spatially controlled nanoparticles designed to colocalize to exposed subendothelial matrices could provide an optimal delivery system for targeted vascular therapeutics. To his end, we aimed to develop the framework for a surface-modified liposomal drug delivery platform designed to preferentially bind collagen type IV. Nontargeted control liposomes (NTL) were formed with bulk DOPC-PEG, 30% cholesterol, and 0.1 mol% Rhodamine-DOPE. DSPE-PEG-DBCO lipids were conjugated to peptides previously shown to bind collagen IV via copper-free click chemistry and inserted at 5 mol% to form collagen-targeting liposomes (CTL), either at hydration (PreCTL) or by postinsertion via micellar transfer (PostCTL). Peptide conjugation was confirmed by matrix-assisted laser desorption/ionization time of flight, and liposomes were characterized by dynamic light scattering and electrophoretic mobility. Liposome binding was assayed on collagen IV matrices dried at 3 μg/cm2 and quantified by fluorescence at 0- to 24-hour static 37°C incubation. All liposome formulations exhibited a narrow size distribution (∼100 nm) and neutral-low positive charge. CTLs demonstrated a significant increase in binding vs NTLs (Fig). CTLs demonstrated significant affinity for collagen IV binding in a static environment compared with NTLs. Future studies aim to optimize the binding capacity of CTLs via further lipid modifications and under flow conditions mimicking vessel wall hemodynamics. Considering the efficacy demonstrated here, CTLs show promise as the framework for a spatially-controlled drug delivery platform for future application in targeted vascular therapeutics.

Background: We have previously defined mechanisms of intimal hyperplasia that could be targets for molecular therapeutics aimed at vascular pathology. However, biocompatible nanocarriers are needed for effective delivery. Cationic liposomes (CLPs) have been demonstrated as effective nanocarriers in vitro. However, in vivo success has been hampered by cytotoxicity. Recently, neutral PEGylated liposomes (PLPs) have been modified with cell-penetrating peptides (CPPs) to enhance cellular uptake. We aim to establish CPP-modified neutral liposomes as viable molecular nanocarriers in vascular smooth muscle cells. Methods: CLPs, PLPs, and CPP-modified PLPs (R8-PLPs) were assembled with short interfering RNA (siRNA) via ethanol injection. Characterization studies determined liposomal morphology, size, and charge. siRNA encapsulation efficiency was measured via RiboGreen assay. Vascular smooth muscle cells were exposed to equal lipid/siRNA across all groups. Rhodamine-labeled liposomes were used to quantify cell association via fluorometry, live/dead dual stain was used to measure cytotoxicity, and gene silencing was measured by quantitative polymerase chain reaction. Results: R8-PLPs exhibited increased encapsulation efficiency equivalent to CLPs. PLPs and R8-PLP-5 mol% and R8-PLP-10 mol% had no cytotoxic effect. CLPs demonstrated significant cytotoxicity. R8-PLP-5 mol% and R8-PLP-10 mol% exhibited increased cell association versus PLPs. R8-PLP-10 mol% resulted in significant gene silencing, in a manner dependent on lipid-to-siRNA load capacity. Conclusions: The negligible cytotoxicity and enhanced cellular association and gene silencing capacity exhibited by R8-PLPs reveal this class of liposomes as a candidate for future applications. Further modifications for optimizing R8-PLPs are still warranted to improve efficacy, and in vivo studies are needed for translational development. However, this could prove to be an optimal nanocarrier for vascular gene therapeutics. (C) 2017 Elsevier Inc. All rights reserved.

Objectives: Vascular intervention results in intimal denudation, exposure of the sub-endothelial matrix, and subsequent intimal hyperplasia (IH), under the control of numerous remodeling mechanisms. Reduction of IH-induced restenosis may be achieved by manipulation of these remodeling pathways through targeted molecular inhibition. Spatially-controlled nanoparticles designed to co-localize to exposed sub-endothelial matrices could provide an optimal delivery system for targeted vascular therapeutics. To his end, we aimed to develop the framework for a surface-modified liposomal drug delivery platform designed to preferentially bind collagen type IV. Methods: Non-targeted control liposomes (NTL) were formed with bulk DOPC-PEG, 30% cholesterol, and 0.1 mol% Rhodamine-DOPE. DSPE-PEG-DBCO lipids were conjugated to peptides previously shown to bind collagen IV via copper-free click chemistry and inserted at 5 mol% to form collagen-targeting liposomes (CTL), either at hydration (PreCTL) or by post-insertion via micellar transfer (PostCTL). Peptide conjugation was confirmed by MALDI-TOF, and liposomes were characterized by DLS and electrophoretic mobility. Liposome binding was assayed on collagen IV matrices dried at 3ug/cm 2 and quantified by fluorescence at 0-24hr static 37°C incubation. Results: All liposome formulations exhibited a narrow size distribution (~100nm) and neutral-low positive charge. CTLs demonstrated a significant increase in binding vs. NTLs (Fig1). Conclusions: CTLs demonstrated significant affinity for collagen IV binding in a static environment compared with NTLs. Future studies aim to optimize the binding capacity of CTLs via further lipid modifications and under flow conditions mimicking vessel wall hemodynamics. Considering the efficacy demonstrated here, CTLs show promise as the framework for a spatially-controlled drug delivery platform for future application in targeted vascular therapeutics.

Objectives: Androgen deficiency (AD) is associated with increased risk of cardio- and peripheral vascular disease, yet the underlying biochemical mechanisms remain unclear. Systemically testosterone (TST) is enzymatically reduced to its more potent metabolite dihydrotestosterone (DHT) or is aromatized to estradiol, which differentially stimulate androgen and estrogen receptor-mediated pathways, respectively. We have previously demonstrated an inverse relationship between TST levels and the cellular processes of intimal hyperplasia (IH) in vitro. Here we investigated TST and DHT replacement in the attenuation of IH in an in vivo model of AD. Methods: Sub- to high physiologic levels of TST or DHT was administered via pellet implants in aged orchiectomized rats (0.5-5mg). Young intact (YI), aged intact (AI), and orchiectomized placebo (Plac) rats served as controls. After 14d hormone replacement rats underwent balloon angioplasty of the left common carotid. 14d post-injury animals were euthanized, systemic hormone levels were determined by ELISA and comparative weight analysis of androgen sensitive organs (Table 1), and carotid intima:media (I:M) was quantified. Results: I:M was decreased in AI animals and with higher physiological TST replacement compared to YI controls (Fig 1). I:M was higher in Plac, sub- and low-physiological TST animals and at all DHT levels. Conclusions: Aging and the normal reduction of TST was protective against IH when compared to young animals. However, pathological AD and sub-physiological hormone replacement increased IH. While physiological TST replacement attenuated this effect, equivalent DHT replacement was not protective, but instead exacerbated the hyperplastic response. Future studies will investigate if the protective effect of physiological TST replacement could be via its conversion to estradiol and downstream estrogen receptor signaling and if estrogen therapy attenuates IH in AD males.

BACKGROUND:Androgen deficiency (AD) is associated with increased risk of vascular disease. Dysfunctional remodeling of the vessel wall and atypical proliferative potential of vascular smooth muscle cells (VSMCs) are fundamental processes in the development of intimal hyperplasia (IH). We have demonstrated an inverse relationship between dihydrotestosterone (DHT) levels, matrix metalloproteinase activity, and VSMC migration and proliferation in vitro. Here, we investigated the role of AD and testosterone (TST) replacement in IH development in an animal model of vascular injury to elucidate mechanisms modulated by AD that could be playing a role in the development of vascular pathogenesis. METHODS:Aged orchiectomized male rats underwent TST supplementation via controlled release pellet (0.5-35 mg). Young adult and middle-age adult intact (MI) and orchiectomized placebo (Plac) groups served as controls. All groups underwent balloon angioplasty of the left common carotid at a 14-d post-TST. Carotid tissue was collected at a 14-d post-balloon angioplasty and subjected to morphologic and immunohistochemical analyses. Human male VSMCs were treated with DHT (0-3000 nM) for 24 h then subjected to quantitative PCR for gene expression analyses and costained for F-actin and G-actin for visualization of cytoskeletal organization. RESULTS:I:M ratio was increased in Plac, subphysiological, low-physiological, and high pharmacologic level TST animals compared with MI controls but was decreased with high-physiological TST supplementation. Injury-induced expression of previously defined matrix metalloproteinase remodeling enzymes was not significantly affected by TST status. Urotensin (UTS) receptor (UTSR) staining was low in injured vessels of all young adult intact, MI, and Plac controls but was significantly upregulated in all groups receiving exogenous TST supplementation, irrespective of dose. In vitro DHT exposure increased the expression of UTSR in VSMCs in a dose-dependent manner. However, this did not correlate with any change in proliferative markers. F:G actin staining revealed that DHT-induced cytoskeletal organization in a dose-dependent manner. CONCLUSIONS:AD increased IH development in response to vascular injury, whereas physiological TST replacement attenuated this effect. AD-induced IH occurs independent of matrix remodeling mechanisms known to be heavily involved in vascular dysfunction, and AD alone does not affect the UTS and/or UTSR mechanism. Exogenous TST and/or DHT increases UTSR pathway signaling in vitro and in vivo. This modulation correlates to a shift in cytoskeletal organization and may exacerbate vasoconstrictive pathogenesis. While physiological TST replacement attenuates AD-modulated IH development, its UTS-mediated effect on vasotone may prove deleterious to overall vascular function.

The use of short interfering RNA (siRNA) to degrade messenger RNA in the cell cytoplasm and transiently attenuate intracellular proteins shows promise in the inhibition of vascular pathogenesis. However, a critical obstacle for therapeutic application is a safe and effective delivery system. Biodegradable polymers are promising alternative molecular carriers for genetic material. Here, we aim to perform a comparative analysis of poly(B-amino ester) (PBAE) and polyethylenimine (PEI) polymers in their efficacy for vascular smooth muscle cell transfection using siRNA against the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene as our test target.Methods: Human aortic smooth muscle cells (HASMC) were transfected in vitro with polymers conjugated to GAPDH or negative control (NC) siRNAs. Increasing siRNA: polymer ratios were tested for optimal transfection efficiency. DharmaFECT2 chemical transfection complexes were used for comparative analysis. Live/dead dual stain was used to measure cell viability, and GAPDH gene silencing was measured by quantitative polymerase chain reaction normalized to 18S.Results: The highest rate of PEI-mediated silencing was achieved with a 9mL polymer: 220 pmol/mL siRNA conjugate (16 +/- 2% expression versus NC; n = 6). Comparable PBAEmediated silencing could be achieved with a 1.95 mu L polymer: 100 pmol/mL siRNA conjugate (10 +/- 1% expression versus NC; n = 5). Transfection using PEIs resulted in silencing equivalent to other methods but with less efficiency and increased cell toxicity at 24h polymer exposure. Decreasing PEI exposure time to 4 h resulted in similar silencing efficacy (21 +/- 9% expression versus NC, n = 6) with an improved toxicity profile.Conclusions: Polymeric bioconjugates transfected HASMCs in a manner similar to chemical complexes, with comparable cell toxicity and silencing efficiency. PEI bioconjugates demonstrated silencing equivalent to PBAE bioconjugates, although less efficient in terms of required polymer concentrations. Given the cost-to-benefit difference between the assayed polymers, and PEI's ability to transfect HASMCs within a short duration of exposure with an improved toxicity profile, this study shows that PEI bioconjugates are a potential transfection agent for vascular tissue. Future studies will expand on this method of gene therapy to validate delivery of gene-specific inhibitors aimed at attenuating smooth muscle cell proliferation, adhesion, and migration. These studies will lay the framework for our future experimental plans to expand on this method of gene therapy for in vivo transfection in animal models of vascular disease. (C) 2015 Elsevier Inc. All rights reserved.

Objectives: Clinical and epidemiological studies have shown low testosterone (TST) is associated with increased risk of vascular disease, yet the molecular mechanisms remain unclear. Matrix metalloproteinases (MMPs) are implicated in dysfunctional remodeling. Our group has previously demonstrated an inverse relationship between TST levels and MMP activity in vitro. We have also demonstrated androgen deficiency (AD)-modulated inflammatory signaling does not influence systemic MMPs in vivo. Here we investigated the role of AD and TST replacement in MMP-modulated remodeling in response to injury. Methods: Sub-physiological and physiological TST replacement was administered via implanted pellets in aged orchiectomized rats (0.5-5mg, Table 1). Serum TST was determined by ELISA. At 14d TST replacement rats underwent balloon angioplasty of the left common carotid. Young intact (YI), aged intact (AI), and orchiectomized placebo (Plac) animals served as controls. Carotids were collected 14d post-injury for intima:media (I:M) and MMP quantification. Results: I:M was increased in Plac, sub-, and low-physiological TST animals compared to AI controls, and decreased with higher physiological TST (Fig 1). Injury-induced expression of MMPs involved in vascular remodeling was not significantly affected by TST status (Table 2). Conclusions: AD is associated with hyperplasia development in response to vascular injury while physiological TST replacement attenuated this effect. This attenuation occurs independent of MMP mechanisms known to be heavily involved in vascular remodeling. Future in vivo studies will examine molecular targets involved in AD and the effect of TST replacement in the acute inflammatory response to vascular injury. These studies are needed in determining if TST replacement therapy in AD men should be evaluated for attenuation of vascular pathogenesis.

Peripheral vascular disease is a proinflammatory-modulated disease processes. Zyflamend is a polyherbal dietary supplement that has been previously shown to have potent anti-inflammatory properties, inhibit cell cycle progression and proliferation, and attenuate inflammatory-modulated pathologies. Here we examined the effect of Zyflamend on inflammatory-dependent vascular remodeling in an animal model of vascular disease. Diets of aged male rats were supplemented with human equivalent doses of Zyflamend or placebo for 7 days prior to balloon angioplasty injury of the right carotid artery and continuously thereafter. Carotid tissue was collected 28 days after injury and stained for intima:media (I:M) and matrix metalloproteinase (MMP) quantification. Serum was analyzed via qualitative antibody array and quantitative multiplex array. I:M was decreased in Zyflamend animals compared with placebo (0.43 ± 0.14 vs 0.82 ± 0.12, n = 10; P < .05). MMPs were downregulated in the tissue of Zyflamend animals compared with placebo. Qualitative screening indicated injury-induced cytokines may be downregulated by Zyflamend. Quantitative analysis is ongoing. Dietary supplementation with Zyflamend decreases IH development in vivo, possibly via MMP regulatory mechanisms under the control of inflammatory signaling cascades. To our knowledge this is the first study to investigate the potential for this polyherbal mixture to modulate vascular pathogenesis. Future studies will evaluate these processes in a larger animal population, and this pilot animal study will serve as the backbone for a clinical trial investigating Zyflamend as a therapeutic anti-inflammatory agent in the prevention of vascular disease progression.

Background: Androgen deficiency (AD) is associated with increased risk of atherosclerosis, cardiovascular, and peripheral arterial disease. Although the biochemical and molecular mechanisms underlying this risk remain unclear, higher testosterone (TST) levels correlate to significant immunoprotective molecular and cellular responses. Our group has previously demonstrated that female sex hormones influence vascular pathogenesis via inflammatory-modulated matrix metalloproteinase (MMP) regulation. Here we investigated the role of AD and androgen replacement therapy in the modulation of these hormonally responsive pathways that could be playing a role in the development of vascular pathogenesis.Methods: Aged orchiectomized male rats underwent TST supplementation per controlled release pellet implantation (0-150 mg). Young and aged intact groups served as controls. Serum was collected at 0-4 wk and analyzed by enzyme-linked immunosorbent assays, qualitative cytokine screening, and quantitative multiplex analyses. Human aortic smooth muscle cells were treated with 4,5 alpha-dihydrotestosterone (DHT; 0-3000 nM) before or after interleukin 1 beta (IL-1 beta; 5 ng/mL) stimulation. Quantitative polymerase chain reaction and ingel zymography was used to assay the effect on MMP expression and activity.Results: Subphysiological, physiological, and supraphysiological levels of TST were achieved with 0.5, 2.5, and 35 mg TST pellet implants in vivo, respectively. Inflammatory arrays indicated that interleukin cytokines, specifically IL-2, IL-6, IL-10, IL-12, and IL-13, were elevated at subphysiological level of TST, whereas TST supplementation decreased interleukins. Supraphysiological TST resulted in a significant increase in MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in vivo. Pretreatment with IL-1b slightly increased membrane type 1-MMP (MT1-MMP) and MMP-2 expression at low to mid-level DHT exposure in vitro, although these trends were not statistically significant.Conclusions: Here we demonstrate AD is a proinflammatory modulator and indicate that MMP-independent mechanisms may play a role downstream of AD-induced inflammatory signaling in dysfunctional vascular remodeling. Future in vivo studies will examine AD and TST supplementation in acute inflammatory response to vascular injury and in MMP-modulated vascular disease. (C) 2014 Elsevier Inc. All rights reserved.

Objectives: Androgen deficiency (AD) is associated with increased risk of vascular disease, yet the molecular mechanisms remain unclear. Our group has previously shown testosterone regulates matrix metalloproteinases (MMP) in a dose-dependent manner in vitro. Here we investigated the role of AD and androgen replacement therapy (ART) on inflammatory cytokines and MMP-modulated intimal hyperplasia (IH) development in vivo. Methods: Aged orchiectomized (AO) rats were implanted with increasing doses of testosterone pellets (TST; 0.5-150mg). ELISA and multiplex array determined serum TST and cytokine levels. Young intact (YI), Aged intact (AI), and AO rats given placebo (Plac) or TST supplementation underwent balloon angioplasty of the left common carotid following 14d ART. Tissue samples were collected 14d post-injury for Intima:Media (I:M) or MMP quantification. Results: Therapeutic TST doses were achieved at 14d with 0.5, 2.5, 5, and 35mg pellets when compared to controls (Table 1). Interleukin family isoforms were elevated at sub-physiological TST levels but returned to control levels with physiological TST (Table 2). I:M was decreased in AI and physiological TST levels compared to YI (Fig 1). I:M was increased with sub- and supra-physiological TST. Injury-induced expression of MMP-2 was highest in AI and physiological TST conditions, though these values were not significant (Table 3). Analysis of other MMP isoforms is ongoing. Conclusions: We demonstrated that low testosterone levels increase interleukin inflammatory signaling, regulate MMP expression, and increase IH development in vivo. This effect is reversed by physiologic testosterone supplementation. AD could be playing a role in vascular disease via MMP regulatory mechanisms under the control of inflammatory signaling cascades. Future studies will examine targeted inhibition of inflammatory-modulated MMP mechanisms in the prevention of dysfunctional vascular remodeling.

Low testosterone levels have been associated with increased risk for vascular disease. Previous studies have shown a significant decrease in intimal hyperplasia (IH) development in the presence of testosterone. Matrix metalloproteinases (MMPs) play a major role in vascular remodeling due to their ability to selectively degrade components of the extracellular matrix, allowing pathological vascular smooth muscle cell (VSMC) migration and proliferation to the intimal layer. Our group has previously shown a positive correlation between estrogen/progesterone and IH development via increased MMP activity in vitro and in vivo. Here we hypothesized that testosterone attenuates the cellular processes of IH by inhibiting MMP expression and activity.

Chapman, Jason R. MD; Mountain, Deidra J.H. PhD; Kirkpatrick, Stacy S. BS; Stevens, Scott L. MD; Arnold, Josh D. MD; Goldman, Mitchell H. MD, FACS; Freeman, Michael B. MD, FACS; Grandas, Oscar H. MD, FACS Author Information