Qualitative investigation of µ- and κ-opioid receptor distribution in the brains of budgerigars (Melopsittacus undulatus).

OBJECTIVE: To perform a qualitative analysis of the distribution of µ- and κ-opioid receptor mRNA in the forebrain and midbrain of budgerigars (Melopsittacus undulatus). SAMPLE: 8 brains of male budgerigars. PROCEDURES: Custom-made RNA hybridization probes (RNAscope; Advanced Cell Diagnostics Inc) were used for fluorescent in situ hybridization (FISH) assays performed on selected fresh frozen prepared sections of brain tissue to identify µ- and κ-opioid receptor mRNA. RESULTS: There was κ-opioid receptor mRNA present in the nucleus dorsomedialis posterior thalami, lateral striatum, mesopallium, tractus corticohabenularis et corticoseptalis, griseum et fibrosum, stratum griseum centrale, medial striatum, and area parahippocampalis. There was µ-opioid receptor mRNA present in the stratum griseum centrale, stratum opticum, dorsomedialis posterior thalami, area parahippocampalis, medial striatum, and nidopallium intermedium. CLINICAL RELEVANCE: Consistent with previous studies in pigeons and domestic chicks, κ-opioid receptors were more abundant than µ-opioid receptors in the samples of the present study. The results of this study may also help explain the hyperexcitability or lack of response that can occur with administration of pure µ-opioid receptor agonists, but not κ-opioid receptor agonists. This study was not quantitative, so further research should endeavor to compare the various regions of the brain using FISH technology.

Liposomal Nanocarriers Designed for Sub-Endothelial Matrix Targeting under Vascular Flow Conditions.

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.

Advances in the Formulation and Assembly of Non-Cationic Lipid Nanoparticles for the Medical Application of Gene 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.

The Efficacy of Systemic Doxycycline Administration as an Inhibitor of Intimal Hyperplasia after Balloon Angioplasty Arterial Injury.

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.

Cell mimetic liposomal nanocarriers for tailored delivery of vascular therapeutics.

Liposomal delivery systems (LDSs) have been at the forefront of medicinal nanotechnology for over three decades. Increasing LDS association to target cells and cargo delivery is crucial to bolstering overall nanodrug efficacy. Our laboratory aims to develop LDSs for molecular therapeutics aimed at vascular pathology. We have previously established a liposome platform that is an effective delivery system for RNA interference in vascular cell types by using polyethylene glycol (PEG) decorated liposomes bearing an octa-arginine (R8) cell penetrating peptide (CPP). Further tailoring liposome membranes to mimic vascular cell membrane lipid constituents may be a promising strategy for increasing cargo delivery. Here we aimed to develop liposomal formulations that could make use of diacylglycerol (DAG) and phosphatidylserine (PS), naturally occurring lipid species that are known to influence vascular cell function, as a facile and efficient means to increase nanodrug efficacy without compromising clinical viability. We investigated the ability of DAG and PS to amplify the cellular uptake of our previously established LDS platform loaded with small interfering ribonucleic acid (siRNA) cargo. Cellular fluorescence microscopy experiments were performed in conjunction with quantitative cell association assays and cytotoxicity assays to analyze the effect of DAG/PS on the differential delivery of fluorescently-tagged liposomes to vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs) and on liposomal-mediated toxicity. In these studies, significant, dose-dependent increases in association to target cells were observed, as well as cell-type specific effects on cell viability. The stability and encapsulation-efficiency of the DAG/PS-modified LDSs were analyzed by standard nanoparticle characterization methods, and siRNA transfection efficacy was quantified to gauge delivery potential as a function of DAG/PS modification. Our results suggest that the signaling lipids tested here imbue our LDS architectures with increased therapeutic potential, without compromising stability, encapsulation efficiency, or biocompatibility, thus presenting a natural strategy to increase nanodrug efficacy and specificity.

Improving the efficacy of liposome-mediated vascular gene therapy via lipid surface modifications.

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.

Testosterone replacement attenuates intimal hyperplasia development in an androgen deficient model of vascular injury.

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.

Comparative analysis of polymers for short interfering RNA delivery in vascular smooth muscle cells.

UNLABELLED: 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 9µL polymer:220 pmol/mL siRNA conjugate (16 ± 2% expression versus NC; n = 6). Comparable PBAE-mediated silencing could be achieved with a 1.95µL 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.

A Zr-based bulk metallic glass for future stent applications: Materials properties, finite element modeling, and in vitro human vascular cell response.

Despite the prevalent use of crystalline alloys in current vascular stent technology, new biomaterials are being actively sought after to improve stent performance. In this study, we demonstrated the potential of a Zr-Al-Fe-Cu bulk metallic glass (BMG) to serve as a candidate stent material. The mechanical properties of the Zr-based BMG, determined under both static and cyclic loadings, were characterized by high strength, which would allow for the design of thinner stent struts to improve stent biocompatibility. Finite element analysis further complemented the experimental results and revealed that a stent made of the Zr-based BMG was more compliant with the beats of a blood vessel, compared with medical 316L stainless steel. The Zr-based BMG was found to be corrosion resistant in a simulated body environment, owing to the presence of a highly stable ZrO2-rich surface passive film. Application-specific biocompatibility studies were conducted using human aortic endothelial cells and smooth muscle cells. The Zr-Al-Fe-Cu BMG was found to support stronger adhesion and faster coverage of endothelial cells and slower growth of smooth muscle cells than 316L stainless steel. These results suggest that the Zr-based BMG could promote re-endothelialization and potentially lower the risk of restenosis, which are critical to improve vascular stent implantation integration. In general, findings in this study raised the curtain for the potential application of BMGs as future candidates for stent applications. STATEMENT OF SIGNIFICANCE: Vascular stents are medical devices typically used to restore the lumen of narrowed or clogged blood vessel. Despite the clinical success of metallic materials in stent-assisted angioplasty, post-surgery complications persist due to the mechanical failures, corrosion, and in-stent restenosis of current stents. To overcome these hurdles, strategies including new designs and surface functionalization have been exercised. In addition, the development of new materials with higher performance and biocompatibility can intrinsically reduce stent failure rates. The present study demonstrates the advantages of a novel material, named bulk metallic glass (BMG), over the benchmarked 316L stainless steel through experimental methods and computational simulations. It raises the curtain of new research endeavors on BMGs as competitive alternatives for stent applications.

Low testosterone elevates interleukin family cytokines in a rodent model: a possible mechanism for the potentiation of vascular disease in androgen-deficient males.

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 4,5α-dihydrotestosterone (DHT; 0-3000 nM) before or after interleukin 1ß (IL-1ß; 5 ng/mL) stimulation. Quantitative polymerase chain reaction and in-gel 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-1ß 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.

Androgens regulate MMPs and the cellular processes of intimal hyperplasia.

BACKGROUND: Testosterone deficiency has been associated with an increased risk of vascular disease. Matrix metalloproteinases (MMPs) have been implicated in vascular remodeling. Our group has demonstrated an association between female hormones and MMP-modulated intimal hyperplasia. In the present study, we investigated testosterone in the modulation of MMPs and the cellular processes of intimal hyperplasia. MATERIALS AND METHODS: Male vascular smooth muscle cells (VSMCs) were treated with a range of testosterone or dihydrotestosterone (DHT) concentrations (0.3-3000 nM). MMPs were assayed using quantitative polymerase chain reaction, Western blot analysis, and zymography. VSMC migration and proliferation were assayed using Boyden chamber and MTT assays. RESULTS: MT1-MMP gene expression was not affected by low DHT exposure but was downregulated at high levels (3000 nM = 85% ± 3%). TIMP-2 gene expression was downregulated at low DHT exposure (0.3 nM = 82% ± 4%, 3.0 nM = 82% ± 1%) but was not affected at high levels. MMP-2 enzymatic activity was increased at low DHT exposure (3.0 nM = 110% ± 4%) and decreased below basal levels at high doses (300 nM = 91% ± 7%, 3000 nM = 77% ± 8%). High concentrations of DHT decreased VSMC migration (3.0 nM = 72% ± 9%, 30 nM = 50% ± 6%, 300 nM = 47% ± 5%, 3000 nM = 53% ± 6%). Testosterone also decreased migration but had less effect. The highest tested concentration of DHT and testosterone decreased the basal VSMC proliferation (3000 nM = 87% ± 3% and 87% ± 4% respectively). CONCLUSIONS: The DHT levels differentially affected the expression of regulatory isoforms responsible for the activation and inhibition of MMP-2, leading to an inverse relationship among the DHT levels, MMP-2 activity, and VSMC migration. In vivo studies will be used to examine testosterone deficiency and supplementation in MMP-modulated intimal hyperplasia in animal models of vascular disease. These studies are needed as a prerequisite to determining whether testosterone replacement in testosterone-deficient men should be evaluated for attenuation of atherosclerosis.

Effect of hormone replacement therapy in matrix metalloproteinase expression and intimal hyperplasia development after vascular injury.

BACKGROUND: Postmenopausal women taking hormone replacement therapy (HRT) require secondary intervention after vascular reconstruction more frequently than women not taking HRT, often due to increased development of intimal hyperplasia (IH). Matrix metalloproteinases (MMPs) play a role in IH by degradation and remodeling of components of the vascular basement membrane. The MMP pathway is regulated by a balance between MMPs, membrane-type MMPs (MT-MMPs), and tissue inhibitor of MMPs (TIMPs). We have recently provided evidence for unbalanced regulation of the MT1-MMP/MMP-2 pathway in vascular smooth muscle cells (VSMCs) exposed to hormones in vitro. Herein we study the role of HRT in the development of IH in a postmenopausal rodent model of vascular injury and in the modulation of this MMP regulatory pathway in vivo. METHODS: Female rats were aged to 12 months. Animals were ovariectomized (OVX) and 4 weeks later hormones or placebo was delivered via a 90-day slow-release pellet. After 6 weeks of HRT each rat underwent balloon angioplasty of the left common carotid artery. At 14 days postinjury tissue samples were collected and stained with trichrome elastin and for isoform-specific MMPs. RESULTS: After vascular injury, the intima:media (I:M) ratio was decreased in OVX rats receiving placebos as compared with non-OVX controls (P < 0.05). In OVX animals receiving HRT, estrogen with and without progesterone and progesterone alone slightly increased I:M ratio compared with placebo, although no significant difference was found in any HRT group. Injury-induced intimal expression of MMP-2 and -9 was decreased in OVX placebo animals compared with non-OVX controls (P < 0.05). MMP-2 and -9 levels were subsequently increased by each type of hormone therapy compared with placebo, with a significant increase in MMP-9 in response to estrogen with and without progesterone (P < 0.05). Conversely, TIMP-2 was decreased by estrogen compared with placebo (P < 0.05). There was no effect on intimal MT1-MMP in any group. CONCLUSIONS: In this study we detected a statistically significant decrease in IH as a result of OVX. Subsequent HRT exposure resulted in increased I:M ratios compared with OVX animals given placebo, although significance was not reached with the doses given. Long-term exogenous exposure may have a more deleterious effect compared with acute exposure and should be examined further. We also demonstrated a significant reduction in MMP-2 and -9 and TIMP-2 in response to OVX. Subsequent hormone exposure resulted in the upregulation of MMP-2 and -9 without a counterregulatory increase in TIMP, indicating that HRT modulates the MMP regulatory pathway in vivo. The data suggest that the lack of hormones after OVX protects against pathologic remodeling in our aged model of disease and that exposure to both natural and exogenous hormones could be a negative risk factor resulting in an exaggerated vascular response to injury. Future studies should focus on in vivo manipulation of unbalanced MMP regulation for prevention of IH in response to HRT and in general. Furthermore, the age-associated difference in response to the presence of natural hormones in young vs aged models should be investigated.

Smooth muscle cell polymeric transfection is an efficient alternative to traditional methods of experimental gene therapy.

BACKGROUND: Gene therapy shows promise in the treatment of vascular disease. However, traditional transfection methods commonly used in the laboratory are poorly translatable to in vivo conditions, primarily due to the immune response to viral vectors, the cellular toxicity of chemical transfection, and the technical impracticality of electroporation. Biodegradable polymers have shown promise as a safe, predictable, and nontoxic alternative, relying on endocytosis of synthetic polymeric carriers, which are bioconjugated to the targeted genetic material of choice. However, to date most of the feasibility studies have been exclusively performed in stem cells. Differentiated cell types would be prime targets for therapeutic gene modulation in the prevention of various disease processes. We aim to establish polymeric transfection as a method for gene therapy in cells of vascular origin. Here we compared the efficiency of polymeric transfection with chemical transfection agents routinely used in a laboratory setting in vascular smooth muscle cells. METHODS: Human aortic smooth muscle cells (HASMC) were transfected with fluorescently labeled GAPDH siRNA or negative control (NC) siRNA. Transfection methods included poly(B-amino ester) polymer (StemFECT) bioconjugates, DharmaFECT2 complexes, and Santa Cruz complexes. Conjugate endocytosis was confirmed by fluorescent microscopy, and GAPDH gene silencing was assayed by qPCR normalized to 18S. RESULTS: Santa Cruz reagent complexes were the least efficient, with the maximum achievable gene silencing using a 9 µL reagent : 70 pmol siRNA/mL complex (59% ± 6%; n = 3). Maximum GADPH gene silencing using DharmaFECT2 was achieved with a 1.5 µL reagent : 100 pmol siRNA/mL complex (19% ± 1% expression versus NC; n = 4). Equivalent silencing was achieved using a comparable StemFECT bioconjugate of 1.3 µL polymer : 100 pmol siRNA/mL (25% ± 3% expression versus NC; n = 4; P = NS versus DharmaFECT2). By increasing the StemFECT bioconjugate to 1.95 µL polymer : 100 pmol siRNA/mL, gene silencing was significantly increased (10% ± 1% expression versus NC; n = 6; P < 0.05 versus DharmaFECT2 and StemFECT 1.3:100). CONCLUSION: HASMCs were efficiently transfected using polymeric bioconjugates in a manner comparable to and exceeding other transfection agents routinely used in vitro. This proof of concept establishes polymeric transfection as a viable method for in vitro investigation of differentiated vascular cells. Future studies will expand on this method of gene therapy for ex vivo transfection of whole vessel segments and in vivo transfection in animal models of vascular disease. Our long-term goal is to deliver molecular inhibitors of genes thought to play a role in intimal hyperplasia, restenosis, and vessel graft failure.

Role of MT1-MMP in estrogen-mediated cellular processes of intimal hyperplasia.

BACKGROUND: Hormone replacement therapy increases intimal hyperplasia (IH) following vascular intervention. Matrix metalloproteinases (MMPs) play a role in IH development. We have shown estrogen up-regulates MT1-MMP expression, a transmembrane protein that activates MMP-2, and increases vascular smooth muscle cell (VSMC) collagen invasion via increased MMP-2 activity. Here we hypothesize inhibition of MT1-MMP will prevent hormonally-stimulated increased MMP-2 activation and the downstream cellular processes of IH pathogenesis. METHODS: VSMCs from a postmenopausal donor were transfected with MT1-MMP or negative control siRNAs, treated with estrogen (Est), analyzed by q-PCR, Western blot, zymography, migration, invasion, and proliferation assays. RESULTS: Est treatment of MT1-MMP silenced cells still resulted in increased MT1-MMP expression (C = 41% ± 4%; Est = 52% ± 2%; P < 0.05). Silencing of MT1-MMP decreased basal MMP-2 activity (nonsilenced = 100%; MT1-silenced = 87% ± 3%; P < 0.05) but had no effect on basal invasion or proliferation. Est treatment of MT1-MMP silenced cells still resulted in increased MMP-2 activity (C = 87% ± 3%; Est = 101% ± 4%; P < 0.05) and invasion (C = 89% ± 6%; Est = 109% ± 3%; P < 0.05) compared with MT1-MMP silenced control cells. However, silencing of MT1-MMP did inhibit Est- and serum-stimulated proliferation (C = 106% ± 18%; Est = 104% ± 16%; FBS = 121% ± 24%; P = NS). CONCLUSION: Silencing of MT1-MMP in aged VSMCs results in impaired but not complete inhibition of basal and Est-stimulated increases in MMP-2 activity. Other mechanisms appear to be playing a role in hormonally-regulated cellular processes of IH pathogenesis. Future studies will target other signaling cascades, with the goal of identifying mechanisms responsible for hormonally-modulated unbalanced MMPs. In vivo manipulation of the expression patterns of MT1-MMP will be examined for the prevention of IH in animal models of vascular disease.

Serum levels of matrix metalloproteinase-2 as a marker of intimal hyperplasia.

BACKGROUND: A primary component in the development of intimal hyperplasia (IH) in response to vascular injury is basement membrane remodeling. Matrix metalloproteinases (MMPs) play a major role in this process by degradation of basement membrane proteins, mainly collagen type IV. Vascular injury initiates an inflammatory cascade with the release of tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta), and C-reactive protein (CRP). We hypothesize serum levels of these elements may serve as biomarkers of the development of IH. METHODS AND RESULTS: At baseline, 2, 7, 10, and 14 days post-balloon angioplasty of the carotid artery, rat tissue samples were stained with Masson trichrome elastin to examine IH. Intima:media ratios (I:M) increased significantly over time postinjury. Serum samples were collected at the time of tissue sampling, and levels of MMP-2, MMP-9, collagen type IV, TNFalpha, IL-1beta, and CRP were assayed using sandwich enzyme-linked immunosorbent assay (ELISA). MMP-2 serum levels at 7, 10, and 14 days postinjury were significantly elevated compared with baseline. Other elements were not significantly elevated. CONCLUSION: Early and persistent elevation in the serum levels of MMP-2 may be a useful biomarker of basement membrane remodeling and the presence of IH.

Effect of hormones on matrix metalloproteinases gene regulation in human aortic smooth muscle cells.

BACKGROUND: Postmenopausal women receiving hormone replacement therapy have more adverse outcomes after vascular reconstructions. Estrogen-binding receptors have been identified on vascular smooth muscle cells (VSMCs), indicating that vascular function may be under direct hormonal control. A key group of enzymes involved in vascular remodeling are matrix metalloproteinases (MMPs). Here we studied the effect of estrogen (Est) and progesterone (Prog) on MMP gene expression in human VSMCs. METHODS AND RESULTS: VSMCs were incubated with Est (5 ng/mL), Prog (50 ng/mL), Est+Prog combination (Est/Prog), and interleukin-1beta (100 U/mL; IL-1beta). Gene array analysis indicated Est+IL-1beta increased the expression of MMP-3. Reverse transcriptase-polymer chain reaction (RT-PCR) analyses revealed MMP-3 mRNA levels were significantly increased by Est/Prog+IL-1beta treatment. However, Western blot and further RT-PCR analyses indicated no change in MMP-3 in response to hormones alone. RT-PCR analyses revealed membrane type 1 (MT1)-MMP mRNA levels, not MMP-2 or tissue inhibitor of MMP (TIMP), were significantly increased by Est/Prog+IL-1beta, and Western blot analyses confirmed a significant increase in MT1-MMP protein in response to Est alone. CONCLUSION: Estrogen and progesterone affect the MMP pathway of VSMCs via isoform specific mechanisms and may lead to unbalanced MMP regulation. Estrogen up-regulates MT1-MMP without a corresponding increase in TIMP-2, known activator and inhibitor of MMP-2, respectively. Additionally, estrogen up-regulates MMP-3 only in the presence of IL-1beta. This differential regulation, combined with case-specific variations in degree of inflammatory response, may explain why some women receiving exogenous hormone therapy at the time of vascular interventions are more susceptible to complications.

Interleukin-1beta-mediated inhibition of the processes of angiogenesis in cardiac microvascular endothelial cells.

Angiogenesis, the formation of new capillaries from preexisting vessels, plays an essential role in revascularization of the myocardium following myocardial infarction (MI). Interleukin-1beta (IL-1beta), a proinflammatory cytokine increased in the heart following MI, is shown to be essential for angiogenesis in the invasiveness of tumor cells, the progression of arthritic conditions and endometriosis, and the promotion of wound healing. Here we studied the steps of angiogenesis in response to IL-1beta in cardiac microvascular endothelial cells (CMECs) and aortic tissue. Cell cycle progression analysis using flow cytometry indicated a G0/G1 phase cell cycle arrest in IL-1beta-stimulated cells. IL-1beta significantly reduced levels of fibrillar actin in the cytoskeleton, a pre-requisite for tube formation, as indicated by phalloidin-FITC staining. Wound healing assays demonstrated IL-1beta prevents cell-to-cell contact formation. On the other hand, vascular endothelial growth factor-D (VEGF-D) initiated restoration of the cell monolayer. IL-1beta significantly inhibited in vitro tube formation as analyzed by three-dimensional collagen matrix assay. Aortic ring assay demonstrated that IL-1beta inhibits basal and VEGF-D-stimulated microvessel sprouting from aortic rings. The data presented here are novel and of significant interest, providing evidence that IL-1beta impedes the process of angiogenesis in myocardial endothelial cells.

Downregulation of VEGF-D expression by interleukin-1beta in cardiac microvascular endothelial cells is mediated by MAPKs and PKCalpha/beta1.

Interleukin-1beta (IL-1beta) is a proinflammatory cytokine increased in the heart following myocardial infarction. Vascular endothelial growth factors (VEGFs) are implicated in angiogenesis due to their involvement in the recruitment and proliferation of endothelial cells. Here we studied expression of VEGFs in response to IL-1beta in rat cardiac microvascular endothelial cells (CMECs) and investigated the signaling pathways involved in the regulation of VEGF-D. cDNA array analysis indicated that IL-1beta modulates the expression of numerous angiogenesis-related genes, notably decreasing the expression of VEGF-D. RT-PCR and Western blot analyses confirmed decreased expression of VEGF-D in response to IL-1beta. IL-1beta decreased the expression of VEGF-C to a lesser extent with no effects on VEGF-A or -B. Inhibition of ERK1/2, JNKs, or PKCalpha/beta1 alone partially inhibited IL-1beta-induced VEGF-D downregulation. Concurrent inhibition of ERK1/2 or JNKs and PKCalpha/beta1 resulted in a synergistic inhibition of IL-1beta-induced decreases in VEGF-D. Inhibition of ERK1/2 partially inhibited IL-1beta-stimulated inactivation of GSK-3beta with no effect on beta-catenin levels. Inhibition of GSK-3beta using SB216763 inhibited basal VEGF-D expression. We conclude that IL-1beta downregulates VEGF-D expression in CMECs via the involvement of ERK1/2, JNKs, and PKCalpha/beta(1). This is the first report to indicate inhibition of VEGF-D gene expression in response to IL-1beta in cardiac microvascular endothelial cells, a cell type of central interest in angiogenesis.

Interleukin-1beta increases expression and activity of matrix metalloproteinase-2 in cardiac microvascular endothelial cells: role of PKCalpha/beta1 and MAPKs.

Matrix metalloproteinases (MMPs), a family of extracellular endopeptidases, are implicated in angiogenesis because of their ability to selectively degrade components of the extracellular matrix. Interleukin-1beta (IL-1beta), increased in the heart post-myocardial infarction (post-MI), plays a protective role in the pathophysiology of left ventricular (LV) remodeling following MI. Here we studied expression of various angiogenic genes affected by IL-1beta in cardiac microvascular endothelial cells (CMECs) and investigated the signaling pathways involved in the regulation of MMP-2. cDNA array analysis of 96 angiogenesis-related genes indicated that IL-1beta modulates the expression of numerous genes, notably increasing the expression of MMP-2, not MMP-9. RT-PCR and Western blot analyses confirmed increased expression of MMP-2 in response to IL-1beta. Gelatin in-gel zymography and Biotrak activity assay demonstrated that IL-1beta increases MMP-2 activity in the conditioned media. IL-1beta activated ERK1/2, JNKs, and protein kinase C (PKC), specifically PKCalpha/beta(1), and inhibition of these cascades partially inhibited IL-1beta-stimulated increases in MMP-2. Inhibition of PKCalpha/beta(1) failed to inhibit ERK1/2. However, concurrent inhibition of PKCalpha/beta(1) and ERK1/2 almost completely inhibited IL-1beta-mediated increases in MMP-2 expression. Inhibition of p38 kinase and nuclear factor-kappaB (NF-kappaB) had no effect. Pretreatment with superoxide dismutase (SOD) mimetic, MnTMPyP, increased MMP-2 protein levels, whereas pretreatment with SOD and catalase mimetic, EUK134, partially inhibited IL-1beta-stimulated increases in MMP-2 protein levels. Exogenous H(2)O(2) significantly increased MMP-2 protein levels, whereas superoxide generation by xanthine/xanthine oxidase had no effect. This in vitro study suggests that IL-1beta modulates expression and activity of MMP-2 in CMECs.