Myoblast-mediated gene therapy improves functional collateralization in chronic cerebral hypoperfusion.

BACKGROUND AND PURPOSE: Direct extracranial-intracranial bypass surgery for treatment of cerebral hemodynamic compromise remains hindered by complications but alternative simple and safe indirect revascularization procedures, such as an encephalomyosynangiosis (EMS), lack hemodynamic efficiency. Here, the myoblast-mediated transfer of angiogenic genes presents an approach for induction of therapeutic collateralization. In this study, we tested the effect of myoblast-mediated delivery of vascular endothelial growth factor-A (VEGF) to the muscle/brain interface of an EMS in a model of chronic cerebral hypoperfusion. METHODS: Permanent unilateral internal carotid artery-occlusion was performed in adult C57/BL6 mice with or without (no EMS) surgical grafting of an EMS followed by implantation of monoclonal mouse myoblasts expressing either VEGF164 or an empty vector (EV). Cerebral hemodynamic impairment, transpial collateralization, angiogenesis, mural cell investment, microvascular permeability, and cortical infarction after ipsilateral stroke were assessed by real-time laser speckle blood flow imaging, 2- and 3-dimensional immunofluorescence and MRI. RESULTS: VEGF-expressing myoblasts improved hemodynamic rescue by day 14 (no EMS 37±21%, EV 42±9%, VEGF 48±12%; P<0.05 for VEGF versus no EMS and versus EV), together with the EMS take rate (VEGF 60%, EV 18.2%; P<0.05) and angiogenesis of mature cortical microvessels below the EMS (P<0.05 for VEGF versus EV). Importantly, functional and morphological results were paralleled by a 25% reduction of cortical infarction after experimental stroke on the side of the EMS. CONCLUSIONS: Myoblast-mediated VEGF supplementation at the target site of an EMS could help overcome the clinical dilemma of poor surgical revascularization results and provide protection from ischemic stroke.

Therapeutic angiogenesis due to balanced single-vector delivery of VEGF and PDGF-BB.

Therapeutic angiogenesis by delivery of vascular growth factors is an attractive strategy for treating debilitating occlusive vascular diseases, yet clinical trials have thus far failed to show efficacy. As a result, limb amputation remains a common outcome for muscle ischemia due to severe atherosclerotic disease, with an overall incidence of 100 per million people in the United States per year. A challenge has been that the angiogenic master regulator vascular endothelial growth factor (VEGF) induces dysfunctional vessels, if expressed outside of a narrow dosage window. We tested the hypothesis that codelivery of platelet-derived growth factor-BB (PDGF-BB), which recruits pericytes, could induce normal angiogenesis in skeletal muscle irrespective of VEGF levels. Coexpression of VEGF and PDGF-BB encoded by separate vectors in different cells or in the same cells only partially corrected aberrant angiogenesis. In marked contrast, coexpression of both factors in every cell at a fixed relative level via a single bicistronic vector led to robust, uniformly normal angiogenesis, even when VEGF expression was high and heterogeneous. Notably, in an ischemic hindlimb model, single-vector expression led to efficient growth of collateral arteries, revascularization, increased blood flow, and reduced tissue damage. Furthermore, these results were confirmed in a clinically applicable gene therapy approach by adenoviral-mediated delivery of the bicistronic vector. We conclude that coordinated expression of VEGF and PDGF-BB via a single vector constitutes a novel strategy for harnessing the potency of VEGF to induce safe and efficacious angiogenesis.

Stimulation of soluble guanylate cyclase reduces experimental dermal fibrosis.

BACKGROUND: Fibrosis and vascular disease are cardinal features of systemic sclerosis (SSc). Stimulators of soluble guanylate cyclase (sGC) are vasoactive drugs that are currently being evaluated in phase III clinical trials for pulmonary arterial hypertension. OBJECTIVE: To study the antifibrotic potency of sGC stimulators. METHODS: The effect of the sGC stimulator BAY 41-2272 on the release of collagen from dermal fibroblasts was examined. The antifibrotic effects of BAY 41-2272 on prevention and regression of fibrosis in bleomycin-induced dermal fibrosis and in Tsk-1 mice were also studied. Telemetric blood pressure studies in conscious mice were used to study potential hypotensive effects of sGC stimulation. RESULTS: sGC stimulation with BAY 41-2272 dose-dependently inhibited collagen release in dermal fibroblasts from patients with SSc and healthy individuals. Furthermore, BAY 41-2272 stopped the development of bleomycin-induced dermal fibrosis and skin fibrosis in Tsk-1 mice, preventing dermal and hypodermal thickening, reducing the numbers of myofibroblasts and reducing the hydroxyproline content. In addition, BAY 41-2272 was highly effective in the treatment of established fibrosis in the modified models of bleomycin-induced skin fibrosis and Tsk-1 mice. Treatment with sGC stimulators was well tolerated. Relevant antifibrotic doses of BAY 41-2272 did not affect systemic blood pressure and heart rate in mice. CONCLUSIONS: These findings demonstrate potent antifibrotic effects and good tolerability of sGC stimulators in various experimental models of SSc. Given their potential vasoactive properties, sGC stimulators may be promising candidates for the dual treatment of fibrosis and vascular disease in SSc.

Balanced single-vector co-delivery of VEGF/PDGF-BB improves functional collateralization in chronic cerebral ischemia.

The myoblast-mediated delivery of angiogenic genes represents a cell-based approach for targeted induction of therapeutic collateralization. Here, we tested the superiority of myoblast-mediated co-delivery of vascular endothelial growth factor-A (VEGF) together with platelet-derived growth factor-BB (PDGF-BB) on transpial collateralization of an indirect encephalomyosynangiosis (EMS) in a model of chronic cerebral ischemia. Mouse myoblasts expressing a reporter gene alone (empty vector), VEGF, PDGF-BB or VEGF and PDGF-BB through a single bi-cistronic vector (VIP) were implanted into the temporalis muscle of an EMS following permanent ipsilateral internal carotid artery occlusion in adult, male C57BL/6N mice. Over 84 days, myoblast engraftment and gene product expression, hemodynamic impairment, transpial collateralization, angiogenesis, pericyte recruitment and post-ischemic neuroprotection were assessed. By day 42, animals that received PDGF-BB in combination with VEGF (VIP) showed superior hemodynamic recovery, EMS collateralization and ischemic protection with improved pericyte recruitment around the parenchymal vessels and EMS collaterals. Also, supplementation of PDGF-BB resulted in a striking astrocytic activation with intrinsic VEGF mobilization in the cortex below the EMS. Our findings suggest that EMS surgery together with myoblast-mediated co-delivery of VEGF/PDGF-BB may have the potential to serve as a novel treatment strategy for augmentation of collateral flow in the chronically hypoperfused brain.

Reevaluation of the role of VEGF-B suggests a restricted role in the revascularization of the ischemic myocardium.

OBJECTIVE: The endogenous role of the VEGF family member vascular endothelial growth factor-B (VEGF-B) in pathological angiogenesis remains unclear. METHODS AND RESULTS: We studied the role of VEGF-B in various models of pathological angiogenesis using mice lacking VEGF-B (VEGF-B(-/-)) or overexpressing VEGF-B(167). After occlusion of the left coronary artery, VEGF-B deficiency impaired vessel growth in the ischemic myocardium whereas, in wild-type mice, VEGF-B(167) overexpression enhanced revascularization of the infarct and ischemic border zone. By contrast, VEGF-B deficiency did not affect vessel growth in the wounded skin, hypoxic lung, ischemic retina, or ischemic limb. Moreover, VEGF-B(167) overexpression failed to enhance vascular growth in the skin or ischemic limb. CONCLUSIONS: VEGF-B appears to have a relatively restricted angiogenic activity in the ischemic heart. These insights might offer novel therapeutic opportunities.

A universal technology for monitoring G-protein-coupled receptor activation in vitro and noninvasively in live animals.

G-protein coupled receptors (GPCRs) are a versatile and ubiquitous family of membrane receptors that transmit extracellular signals to mammalian cells and constitute the most important class of drug targets. Yet, sensitive and specific methods are lacking that would allow quantitative comparisons of pharmacologic properties of these receptors in physiological or pathological settings in live animals. We sought to overcome these limitations by employing low affinity, reversible beta-galactosidase complementation to quantify GPCR activation via interaction with beta-arrestin. A panel of cell lines was engineered expressing different GPCRs together with the reporter system. In vitro evaluation revealed highly sensitive, dynamic, and specific assessment of GPCR agonists and antagonists. Following implantation of the cells into mice, it was possible for the first time to monitor pharmacological GPCR activation and inhibition in their physiological context by noninvasive bioluminescence imaging in living animals. This technology has unique advantages that enable novel applications in the functional investigation of GPCR modulation in live animals in biological research and drug discovery.

Localization of vascular response to VEGF is not dependent on heparin binding.

The major vascular endothelial growth factor (VEGF) isoforms are splice variants from a single gene that differ in their extent of heparin affinity due to the absence of the heparin binding domain in the smallest isoform (mouse VEGF120, human VEGF121). A long-held assumption that has guided the use of VEGF isoforms clinically has been that their differences in heparin binding dictate their ability to diffuse through tissue, with VEGF121 moving most freely and that the distribution of recombinant VEGF would have therapeutically relevant consequences. To test this assumption, we delivered the genes encoding these isoforms by myoblast-mediated gene transfer, a means of delivering genes to highly localized sites within muscle. Surprisingly, all isoforms induced comparable extremely localized physiological effects. Significantly, irrespective of the isoform delivered, the vessels passing within several micrometers of muscle fibers expressing VEGF displayed sharply delineated changes in morphology. The induction of capillary wrapping around VEGF-producing fibers, and of vascular malformations in the muscle at high levels, did not differ among isoforms. These results indicate that heparin binding is not essential for the localization of VEGF in adult tissue and suggest that the preferential delivery of VEGF121 cDNA for clinical applications may not have a physiological basis.

Collagen matrices enhance survival of transplanted cardiomyoblasts and contribute to functional improvement of ischemic rat hearts.

BACKGROUND: Cardiac cell transplantation is limited by poor graft viability. We aimed to enhance the survival of transplanted cardiomyoblasts using growth factor-supplemented collagen matrices. METHODS AND RESULTS: H9c2 cardiomyoblasts were lentivirally transduced to express firefly luciferase and green fluorescent protein (GFP). Lewis rats underwent ligation of the left anterior descending artery (LAD) ligation to induce an anterior wall myocardial infarction. Hearts (n=9/group) were harvested and restored ex vivo with 1 x 10(6) genetically labeled H9c2 cells either in (1) saline-suspension, or seeded onto (2) collagen-matrix (Gelfoam [GF];), (3) GF/Matrigel (GF/MG), (4) GF/MG/VEGF (10 microg/mL), or (5) GF/MG/FGF (10 microg/mL). Hearts were then abdominally transplanted into syngeneic recipients (working heart model). Controls (n=6/group) underwent infarction followed by GF implantation or saline injection. Cell survival was evaluated using optical bioluminescence on days 1, 5, 8, 14, and 28 postoperatively. At 4 weeks, fractional shortening and ejection fraction were determined using echocardiography and magnetic resonance imaging, respectively. Graft characteristics were assessed by immunohistology. Bioluminescence signals on days 5, 8, and 14 were higher for GF-based grafts compared with plain H9c2 injections (P<0.03). Signals were higher for GF/MG grafts compared with GF alone (P<0.02). GFP-positive, spindle-shaped H9c2 cells were found integrated in the infarct border zones at day 28. Left ventricular (LV) function of hearts implanted with collagen-based grafts was better compared with controls (P<0.05). Vascular endothelial growth factor or fibroblast growth factor did not further improve graft survival or heart function. CONCLUSIONS: Collagen matrices enhance early survival of H9c2 cardiomyoblasts after transplantation into ischemic hearts and lead to improved LV function. Further optimization of the graft design should make restoration of large myocardial infarctions by tissue engineering approaches effective.

Adenoviral human BCL-2 transgene expression attenuates early donor cell death after cardiomyoblast transplantation into ischemic rat hearts.

BACKGROUND: Cell transplantation for myocardial repair is limited by early cell death. Gene therapy with human Bcl-2 (hBcl-2) has been shown to attenuate apoptosis in the experimental setting. Therefore, we studied the potential benefit of hBcl-2 transgene expression on the survival of cardiomyoblast grafts in ischemic rat hearts. METHODS AND RESULTS: H9c2 rat cardiomyoblasts were genetically modified to express both firefly luciferase and green fluorescent protein (mH9c2). The cells were then transduced with adenovirus carrying hBcl-2 (AdCMVhBcl-2/mH9c2). Lewis rats underwent ligation of the left anterior descending artery (LAD) to induce a sizable left ventricular (LV) infarct. Hearts were explanted and the infarcted region was restored using collagen matrix (CM) seeded with 1x10(6) mH9c2 cells (n=9) or AdCMVhBcl-2/mH9c2 cells (n=9). Control animals received CM alone (n=6) or no infarct (n=6). Restored hearts were transplanted into the abdomen of syngeneic recipients in a "working heart" model. Cell survival was evaluated using optical bioluminescence imaging on days 1, 5, 8, 14, and 28 after surgery. The left heart function was assessed 4 weeks postoperatively using echocardiography and magnetic resonance imaging. During 4 weeks after surgery, the optical imaging signal for the AdCMVhBCL2/mH9c2 group was significantly (P<0.05) higher than that of the mH9c2-control group. Both grafts led to better fractional shortening (AdCMVhBcl-2/mH9c2: 0.21+/-0.03; mH9c2: 0.21+/-0.04; control: 0.15+/-0.03; P=0.04) and ejection fraction (AdCMVhBcl-2/mH9c2: 47.0+/-6.2; mH9c2: 48.7+/-6.1; control: 34.3+/-6.0; P=0.02) compared with controls. Importantly, no malignant cells were found in postmortem histology. CONCLUSIONS: Transduction of mH9c2 cardiomyoblasts with AdCMVhBcl-2 increased graft survival in ischemic rat myocardium without causing malignancies. Both AdCMVhBcl-2/mH9c2 and mH9c2 grafts improved LV function.

An angiogenic role for the human peptide antibiotic LL-37/hCAP-18.

Antimicrobial peptides are effector molecules of the innate immune system and contribute to host defense and regulation of inflammation. The human cathelicidin antimicrobial peptide LL-37/hCAP-18 is expressed in leukocytes and epithelial cells and secreted into wound and airway surface fluid. Here we show that LL-37 induces angiogenesis mediated by formyl peptide receptor-like 1 expressed on endothelial cells. Application of LL-37 resulted in neovascularization in the chorioallantoic membrane assay and in a rabbit model of hind-limb ischemia. The peptide directly activates endothelial cells, resulting in increased proliferation and formation of vessel-like structures in cultivated endothelial cells. Decreased vascularization during wound repair in mice deficient for CRAMP, the murine homologue of LL-37/hCAP-18, shows that cathelicidin-mediated angiogenesis is important for cutaneous wound neovascularization in vivo. Taken together, these findings demonstrate that LL-37/hCAP-18 is a multifunctional antimicrobial peptide with a central role in innate immunity by linking host defense and inflammation with angiogenesis and arteriogenesis.

Microenvironmental VEGF distribution is critical for stable and functional vessel growth in ischemia.

The critical role of vascular endothelial growth factor (VEGF) expression levels in developmental angiogenesis is well established. Nonetheless, the effects of different local (microenvironmental) VEGF concentrations in ischemia have not been studied in the adult organism, and VEGF delivery to patients has been disappointing. Here, we demonstrate the existence of both lower and upper threshold levels of microenvironmental VEGF concentrations for the induction of therapeutic vessel growth in ischemia. In the ischemic hind limb, implantation of myoblasts transduced to express VEGF164 at different levels per cell increased blood flow only moderately, and vascular leakage and aberrant preangiomatous vessels were always induced. When the same total dose was uniformly distributed by implanting a monoclonal population derived from a single VEGF-expressing myoblast, blood flow was fully restored to nonischemic levels, collateral growth was induced, and ischemic damage was prevented. Hemangiomas were avoided and only normal, pericyte-covered vessels were induced persisting over 15 mo. Surprisingly, clones uniformly expressing either lower or higher VEGF levels failed to provide any functional benefit. A biphasic effect of VEGF dose on vessel number and diameter was found. Blood flow was only improved if vessels were increased both in size and in number. Microenvironmental VEGF concentrations determine efficacy and safety in a therapeutic setting.

Pharmacodynamics, Pharmacokinetics, and Antiviral Activity of BAY 81-8781, a Novel NF-κB Inhibiting Anti-influenza Drug.

Influenza is a respiratory disease that causes annual epidemics. Antiviral treatment options targeting the virus exist, but their efficiency is limited and influenza virus strains easily develop resistance. Thus, new treatment strategies are urgently needed. In the present study, we investigated the anti-influenza virus properties of D,L-lysine acetylsalicylate ⋅ glycine (BAY 81-8781; LASAG) that is approved as Aspirin i.v. for intravenous application. Instead of targeting the virus directly BAY 81-8781 inhibits the activation of the NF-κB pathway, which is required for efficient influenza virus propagation. Using highly pathogenic avian influenza virus strains we could demonstrate that BAY 81-8781 was able to control influenza virus infection in vitro. In the mouse infection model, inhalation of BAY 81-8781 resulted in reduced lung virus titers and protection of mice from lethal infection. Pharmacological studies demonstrated that the oral route of administration was not suitable to reach the sufficient concentrations of BAY 81-8781 for a successful antiviral effect in the lung. BAY 81-8781 treatment of mice infected with influenza virus started as late as 48 h after infection was still effective in protecting 50% of the animals from death. In summary, the data represent a successful proof of the novel innovative antiviral concept of targeting a host cell signaling pathway that is required for viral propagation instead of viral structures.

Overexpression of dimethylarginine dimethylaminohydrolase reduces tissue asymmetric dimethylarginine levels and enhances angiogenesis.

BACKGROUND: This study was designed to determine whether overexpression of the enzyme dimethylarginine dimethylaminohydrolase (DDAH) could enhance angiogenesis by reducing levels of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA). METHODS AND RESULTS: In DDAH1 transgenic (TG) and wild-type mice (each n=42), the role of DDAH overexpression on angiogenesis was studied by use of the disk angiogenesis system and a murine model of hindlimb ischemia (each n=21). After surgery, animals were treated with either PBS or the NOS inhibitors ADMA or N(omega)-nitro-L-arginine methyl ester (L-NAME; each 250 micromol x kg(-1) x d(-1)) by use of osmotic minipumps (each n=7). L-NAME was chosen to study an inhibitor that is not degraded by DDAH. Neovascularization in the disk angiogenesis system was impaired by both NOS inhibitors; however, TG animals were resistant to the effects of ADMA on neovascularization. Similarly, TG mice were more resistant to the inhibitory effect of ADMA on angioadaptation (angiogenesis and arteriogenesis) after hindlimb ischemia, as assessed by fluorescent microsphere studies and postmortem microangiograms. Enhanced neovascularization and limb perfusion in TG mice were associated with reduced plasma and tissue ADMA levels and enhanced tissue NOS enzyme activity. CONCLUSIONS: We describe a novel mechanism by which DDAH regulates postnatal neovascularization. Therapeutic manipulation of DDAH expression or activity may represent a novel approach to restore tissue perfusion.

Selective pressure-regulated retroinfusion of fibroblast growth factor-2 into the coronary vein enhances regional myocardial blood flow and function in pigs with chronic myocardial ischemia.

OBJECTIVES: We sought to improve regional myocardial delivery and subsequent collateral perfusion induced by basic fibroblast growth factor-2 (FGF-2) using selective pressure-regulated retroinfusion of coronary veins for delivery. This hypothesis was tested in a newly developed pig model with percutaneous induction of chronic ischemia. BACKGROUND: Selective pressure-regulated retroinfusion of coronary veins is a catheter-based procedure that has been shown to provide effective regional delivery of drugs and gene vectors into ischemic myocardium. METHODS: A high-grade stenosis with subsequent progression to total occlusion within 28 days was induced by implanting a reduction stent graft into the left anterior descending artery (LAD). After seven days, a 30-min retroinfusion (anterior cardiac vein) was performed with (n = 7) or without (n = 7) 150 microg FGF-2 and compared with a 30-min antegrade infusion of 150 microg FGF-2 into the LAD (n = 7). Sonomicrometry to assess regional myocardial function at rest and during pacing, and microspheres to assess regional myocardial blood flow, were performed 28 days after implantation of the reduction stent. RESULTS: Retroinfusion of FGF-2 compared favorably with controls and with antegrade infusion of FGF-2 with regard to regional myocardial function at rest (18.5 +/- 4.1% vs. 5.7 +/- 2.9% vs. 7.9 +/- 1.8%, respectively, p < 0.05) and during pacing. Regional myocardial blood flow was also higher in the LAD territory after retroinfusion of FGF-2 (1.07 +/- 0.14 vs. 0.66 +/- 0.07 vs. 0.72 +/- 0.17 ml x min(-1) x g(-1), p < 0.05). CONCLUSIONS: Selective pressure-regulated retroinfusion increased tissue binding of FGF-2 and enhanced functionally relevant collateral perfusion compared with antegrade intracoronary delivery in pigs with chronic myocardial ischemia.

Myoblast-mediated gene transfer for therapeutic angiogenesis and arteriogenesis.

Therapeutic angiogenesis aims at generating new blood vessels by delivering growth factors such as VEGF and FGF. Clinical trials are underway in patients with peripheral vascular and coronary heart disease. However, increasing evidence indicates that the new vasculature needs to be stabilized to avoid deleterious effects such as edema and hemangioma formation. Moreover, a major challenge is to induce new vessels that persist following cessation of the angiogenic stimulus. Mature vessels may be generated by modulating timing and dosage of growth factor expression, or by combination of 'growth' factors with 'maturation' factors like PDGF-BB, angiopoietin-1 or TGF-beta. Myoblast-mediated gene transfer has unique characteristics that make it a useful tool for studying promising novel approaches to therapeutic angiogenesis. It affords robust and long-lasting expression, and can be considered as a relatively rapid form of 'adult transgenesis' in muscle. The combined insertion of different gene constructs into single myoblasts and their progeny allows the simultaneous expression of different 'growth' and 'maturation' factors within the same cell in vivo. The additional insertion of a reporter gene makes it possible to analyze the phenotype of the vessels surrounding the transgenic muscle fibers into which the myoblasts have fused. The effects of timing and duration of gene expression can be studied by using tetracycline-inducible constructs, and dosage effects by selecting subpopulations consistently expressing distinct levels of growth factors. Finally, the autologous cell-based approach using transduced myoblasts could be an alternative gene delivery system for therapeutic angiogenesis in patients, avoiding the toxicities seen with some viral vectors.

Therapeutic angiogenesis/arteriogenesis in the chronic ischemic rabbit hindlimb: effect of venous basic fibroblast growth factor retroinfusion.

Therapeutic induction of angiogenesis has been shown in experimental hindlimb ischemia. An alternative to targeting the ischemic hindlimb tissue via the severely stenosed or occluded artery consists in the intact venous system, e.g., by retroinfusion. We tested whether basic fibroblast growth factor (bFGF) enhances angiogenesis induction. Therefore, we applied bFGF retrogradely as compared to intramuscular application. Furthermore, we assessed whether bFGF-induced angiogenesis was enhanced by low-dose VEGF coapplication. Chronic hindlimb ischemia in rabbits was established by excision of the femoral artery at day 0 (d0). At d7, baseline collateral number in the ischemic limb and collateral flow velocity of contrast agent (frame count score) were assessed. Thereafter, saline solution (control group) or bFGF (20 microg/kg) with or without VEGF (10 microg/kg) was retroinfused through the femoral vein. Alternatively, bFGF (20 microg/kg) was injected into thigh and calf muscles. At d35, collateral growth and flow velocity were quantified, and tissue samples were analyzed for capillary density. In the untreated control group, capillary/muscle fiber (C/FM) ratio of the ischemic limb was 0.87 +/- 0.12, and collateral number as well as frame count score at -d35 did not change compared to d7 (107% +/- 7% and 109% +/- 10% of baseline, respectively). Retrograde application of bFGF induced capillary and collateral growth (C/FM ratio 1.56 +/- 0.19 and frame count 161% +/- 29% of baseline), resulting in enhanced flow velocity (143% +/- 13%), similar to the intramuscular application of bFGF. Additional low-dose VEGF retroinfusion did not further increase capillary/collateral growth (1.49 +/- 0.08 and 172% +/- 26%) nor perfusion velocity (149% +/- 7%). The authors conclude that bFGF retroinfusion is a feasible approach of inducing angiogenesis and arteriogenesis in an ischemic hindlimb, resulting in increased blood perfusion, which was not further extended by additional low-dose VEGF coapplication.

Thrombin has biphasic effects on the nitric oxide-cGMP pathway in endothelial cells and contributes to experimental pulmonary hypertension.

BACKGROUND: A potential role for coagulation factors in pulmonary arterial hypertension has been recently described, but the mechanism of action is currently not known. Here, we investigated the interactions between thrombin and the nitric oxide-cGMP pathway in pulmonary endothelial cells and experimental pulmonary hypertension. PRINCIPAL FINDINGS: Chronic treatment with the selective thrombin inhibitor melagatran (0.9 mg/kg daily via implanted minipumps) reduced right ventricular hypertrophy in the rat monocrotaline model of experimental pulmonary hypertension. In vitro, thrombin was found to have biphasic effects on key regulators of the nitric oxide-cGMP pathway in endothelial cells (HUVECs). Acute thrombin stimulation led to increased expression of the cGMP-elevating factors endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) subunits, leading to increased cGMP levels. By contrast, prolonged exposition of pulmonary endothelial cells to thrombin revealed a characteristic pattern of differential expression of the key regulators of the nitric oxide-cGMP pathway, in which specifically the factors contributing to cGMP elevation (eNOS and sGC) were reduced and the cGMP-hydrolyzing PDE5 was elevated (qPCR and Western blot). In line with the differential expression of key regulators of the nitric oxide-cGMP pathway, a reduction of cGMP by prolonged thrombin stimulation was found. The effects of prolonged thrombin exposure were confirmed in endothelial cells of pulmonary origin (HPAECs and HPMECs). Similar effects could be induced by activation of protease-activated receptor-1 (PAR-1). CONCLUSION: These findings suggest a link between thrombin generation and cGMP depletion in lung endothelial cells through negative regulation of the nitric oxide-cGMP pathway, possibly mediated via PAR-1, which could be of relevance in pulmonary arterial hypertension.

Myoblast-mediated gene therapy via encephalomyosynangiosis--a novel strategy for local delivery of gene products to the brain surface.

An encephalomyosynangiosis (EMS) is a temporal muscle graft that is placed onto the surface of the brain to serve as a source for collateral vessel growth for brain revascularization in patients with Moyamoya Disease (MMD). To facilitate an EMS in patients with occlusive cerebrovascular diseases other than MMD, the transfer of pro-angiogenic genes via transplantation of retrovirally transduced myoblasts into the temporal muscle may represent an innovative approach to augment collateralization. Thus, we tested whether retrovirally transfected myoblasts can spontaneously fuse with the non-ischemic and uninjured muscle tissue and if a reporter gene can be stably expressed within the temporal muscle of the EMS. Primary mouse myoblasts expressing a reporter gene were implanted into the temporal muscle prior to an EMS being performed on C57/BL6 mice. Three different implantation modalities were evaluated: (a) intramuscular injection, (b) application of a cell pellet and (c) a combination of both techniques. Myoblast implantation resulted in spontaneous fusion with the host muscle fibers and stable reporter gene expression at both the muscle/brain interface and within the non-ischemic and uninjured temporal muscle in all animals. The mean number of fused hybrid myofibers was 59±28 after injection, 37±30 after pellet application and 60±23 after a combination of both techniques. Regardless of the implantation modality, an abundant extracellular expression of the reporter gene was evident at the muscle/brain interface; in the case of myoblast delivery by injection, expression was also observed around the needle tract marking the implantation site. This method could be used in the future to deliver angiogenic growth factors to the muscle/brain interface in order to improve revascularization after an EMS.

A role for coagulation factor Xa in experimental pulmonary arterial hypertension.

AIMS: Anticoagulation with warfarin is recommended for the treatment of patients with pulmonary arterial hypertension (PAH). However, the therapeutic benefit of anticoagulation has not yet been demonstrated experimentally or clinically. Here, rivaroxaban, an oral, direct factor Xa (FXa) inhibitor, was compared with warfarin and enoxaparin in the prevention of right ventricular (RV) dysfunction and hypertrophy in the monocrotaline (MCT) model of pulmonary hypertension. METHODS AND RESULTS: Sprague-Dawley rats (n = 10 per group) were randomized to receive rivaroxaban, warfarin, enoxaparin, or placebo before receiving a subcutaneous injection of MCT 60 mg/kg or saline. Rivaroxaban and enoxaparin were administered for 28 days starting 4 h before MCT injection; warfarin was given for 35 days initiated 7 days before MCT injection. RV haemodynamics and hypertrophy were assessed 28 days after MCT administration. Rivaroxaban dose-dependently reduced systolic and end-diastolic RV pressure increase and RV hypertrophy. Warfarin reduced RV pressure increase only. Enoxaparin had no effect on either parameter. Severe bleeding occurred in four and five rats treated with warfarin and enoxaparin, respectively, whereas no overt bleeding was observed in rats treated with rivaroxaban. CONCLUSION: Selective, direct inhibition of FXa by rivaroxaban effectively prevented RV dysfunction and hypertrophy in MCT-injected rats, indicating a role for coagulation factors in experimental pulmonary hypertension. Clinical investigation of the impact of early and continued administration of a specific FXa inhibitor such as rivaroxaban on the course of PAH should be considered.

Imaging beta-galactosidase activity in vivo using sequential reporter-enzyme luminescence.

Bioluminescence using the reporter enzyme firefly luciferase (Fluc) and the substrate luciferin enables non-invasive optical imaging of living animals with extremely high sensitivity. This type of analysis enables studies of gene expression, tumor growth, and cell migration over time in live animals that were previously not possible. However, a major limitation of this system is that Fluc activity is restricted to the intracellular environment, which precludes important applications of in vivo imaging such as antibody labeling, or serum protein monitoring. In order to expand the application of bioluminescence imaging to other enzymes, we characterized a sequential reporter-enzyme luminescence (SRL) technology for the in vivo detection of beta-galactosidase (beta-gal) activity. The substrate is a "caged" D-luciferin conjugate that must first be cleaved by beta-gal before it can be catalyzed by Fluc in the final, light-emitting step. Hence, luminescence is dependent on and correlates with beta-gal activity. A variety of experiments were performed in order to validate the system and explore potential new applications. We were able to visualize non-invasively over time constitutive beta-gal activity in engineered cells, as well as inducible tissue-specific beta-gal expression in transgenic mice. Since beta-gal, unlike Fluc, retains full activity outside of cells, we were able to show that antibodies conjugated to the recombinant beta-gal enzyme could be used to detect and localize endogenous cells and extracellular antigens in vivo. In addition, we developed a low-affinity beta-gal complementation system that enables inducible, reversible protein interactions to be monitored in real time in vivo, for example, sequential responses to agonists and antagonists of G-protein-coupled receptors (GPCRs). Thus, using SRL, the exquisite luminescent properties of Fluc can be combined with the advantages of another enzyme. Other substrates have been described that extend the scope to endogenous enzymes, such as cytochromes or caspases, potentially enabling additional unprecedented applications.