Posttransplant Vasculopathy Research Articles

Janus Kinase 3, a Novel Regulator for Smooth Muscle Proliferation and Vascular Remodeling.

Vascular remodeling because of smooth muscle cell (SMC) proliferation is a common process occurring in several vascular diseases, such as atherosclerosis, aortic aneurysm, post-transplant vasculopathy, restenosis after angioplasty, etc. The molecular mechanism underlying SMC proliferation, however, is not completely understood. The objective of this study is to determine the role and mechanism of Janus kinase 3 (JAK3) in vascular remodeling and SMC proliferation. Platelet-derived growth factor-BB, an SMC mitogen, induces JAK3 expression and phosphorylation while stimulating SMC proliferation. Janex-1, a specific inhibitor of JAK3, or knockdown of JAK3 by short hairpin RNA, inhibits the SMC proliferation. Conversely, ectopic expression of JAK3 promotes SMC proliferation. Mechanistically, JAK3 promotes the phosphorylation of signal transducer and activator of transcription 3 and c-Jun N-terminal kinase in SMC, 2 signaling pathways known to be critical for SMC proliferation and vascular remodeling. Blockade of these 2 signaling pathways by their inhibitors impeded the JAK3-mediated SMC proliferation. In vivo, knockdown of JAK3 attenuates injury-induced neointima formation with attenuated neointimal SMC proliferation. Knockdown of JAK3 also induces neointimal SMC apoptosis in rat carotid artery balloon injury model. Our results demonstrate that JAK3 mediates SMC proliferation and survival during injury-induced vascular remodeling, which provides a potential therapeutic target for preventing neointimal hyperplasia in proliferative vascular diseases.

Abstract 521: Janus Kinase 3 is a Novel Regulator for Smooth Muscle Proliferation and Vascularremodeling

Vascular remodeling due to smooth muscle cell (SMC) proliferation is a common process occurring in a number of vascular diseases such as atherosclerosis, aortic aneurysm, posttransplant vasculopathy, and restenosis after angioplasty, etc. The molecular mechanism underlying SMC proliferation, however, is not completed understood. Our present study has identified Janus kinase 3 (JAK3), a member of the Janus kinase family, as a novel regulator for SMC proliferation. Platelet-derived growth factor (PDGF)-BB, a SMC mitogen, induces JAK3 expression and phosphorylation while stimulating SMC proliferation. Janex-1, a specific inhibitor of JAK3, or knockdown of JAK3 by shRNA inhibits SMC proliferation. Conversely, ectopic expression of JAK3 promotes SMC proliferation. Interestingly, JAK3 does not affect SMC contractile protein expression, suggesting that JAK3 mediate the proliferation, but not the phenotypic modulation of SMC. Mechanistically, JAK3 promotes SMC proliferation by regulating phosphorylation of Signal transducer and activator of transcription 3 and c-Jun N-terminal kinase. In vivo, by using a rat carotid balloon-injury model, we found that knockdown of JAK3 significantly attenuates injury-induced neointima formation. Importantly, JAK3 knockdown blocked the expression of proliferating cell nuclear antigen, suggesting that JAK3 is essential for SMC proliferation in vivo. Moreover, JAK3 knockdown prompts apoptosis in neointima SMC, indicating that JAK3 also stimulates SMC survival during neointima formation. Collectively, our data demonstrate that JAK3 mediates vascular remodeling by promoting both the SMC proliferation and survival.

Synergistic Therapeutic Vascular Cytoprotection against Complement-Mediated Injury Induced via a PKCα-, AMPK-, and CREB-Dependent Pathway

Endothelial injury and dysfunction precede accelerated arterial disease in allograft vasculopathy and systemic autoimmune diseases and involve pathogenic Abs and complement. Recent reports suggest that switching to rapamycin from calcineurin antagonists reduces posttransplant vasculopathy and prolongs survival following cardiac transplantion. The majority of these patients also receive statin therapy. We examined potential mechanisms underlying this protective response in human endothelial cells and identified synergy between rapamycin and atorvastatin. Mechanistically, atorvastatin and rapamycin activated a protein kinase Cα, AMP-activated kinase, and CREB-dependent vasculoprotective pathway, which induced decay-accelerating factor (DAF) promoter activity via binding to the cAMP response element, mutation of which attenuated promoter activity. This response significantly increased endothelial cell surface DAF and enhanced protection against complement-mediated injury. Synergy with rapamycin was reproduced by simvastatin, whereas combining atorvastatin with cyclosporine or mycophenolate in place of rapamycin was ineffective. Importantly, synergy was reproduced in vivo, in which only atorvastatin and rapamycin therapy in combination was sufficient to induce DAF on murine aortic endothelium. We believe this pathway represents an important therapeutically inducible vasculoprotective mechanism for diseases mediated by pathogenic Abs and complement, including posttransplant vasculopathy and systemic lupus erythematosus. Although our study focuses on the vascular endothelium, the findings are likely to be broadly applicable, given the diverse cellular expression of DAF.

Modulation of Smooth Muscle Cell Phenotype

The causes of vascular diseases are complex and strongly influenced by environmental factors as well as genetic predisposition. Ultimately, these factors alter the structure and/or functional properties of the arterial wall disrupting vascular homeostasis. Vascular smooth muscle cells (SMCs) have been implicated in the pathogenesis of vascular diseases including atherosclerosis, systemic and pulmonary hypertension, aortic aneurysm and dissection, posttransplant vasculopathy, and restenosis after percutaneous coronary intervention.1 In response to arterial injury and the release and activation of growth factors, contractile SMCs populating the tunica media of the arterial wall downregulate a set of genes encoding SMC-restricted contractile proteins and concomitantly upregulate genes involved in the secretion of extracellular matrix, cell migration, adhesion, and proliferation.1,2 Comprehensive understanding of the molecular programs regulating modulation of SMC phenotype will be necessary to develop preventive and targeted therapeutic strategies for vascular disease. Article, see p 685 The MADS box transcription factor, serum response factor (SRF), and the SMC lineage-restricted transcriptional coactivator, myocardin, lie at the center of a molecular program regulating SMC differentiation and the contractile SMC phenotype.2–4 Most but not all genes encoding SMC contractile proteins contain CArG boxes, or SRF binding sites, in their promoter and/or transcriptional enhancers.3,4 In response to a variety of intracellular signals including Rho/ROCK, MAP kinase, and calcium, myocardin associates with SRF and the myocardin/SRF complex binds to CArG boxes activating transcription of SMC contractile genes.1,4 Remarkably, forced expression of myocardin in embryonic stem cells is necessary and sufficient to activate endogenous SMC genes.5 Moreover, vascular SMCs are not observed in the aorta Myocd −/− null embryos.6 Ablation of the Myocd gene in neural crest–derived SMCs populating the great arteries causes a cell autonomous block in expression of genes encoding SMC contractile proteins. …

Heme oxygenase-1 expression enhances vascular endothelial resistance to complement-mediated injury through induction of decay-accelerating factor: a role for increased bilirubin and ferritin

AbstractCatabolism of free heme by heme oxygenase-1 (HO-1) generates carbon monoxide, biliverdin, and free iron (Fe). These end-products are responsible for much of the biologic activity of HO-1, including anti-inflammatory, antiapo-ptotic, antiproliferative, and antioxidant effects. We have identified an additional cytoprotective action, the regulation of complement activation, mediated via induction of decay-accelerating factor (DAF). Pharmacologic inhibition or short-interfering RNA (siRNA) depletion of HO-1 prevented induction of DAF expression in human endothelial cells. In contrast, HO-1 agonists hemin and cobalt protoporphyrin IX significantly increased DAF protein expression, reflecting an increase in transcription and steady-state mRNA. Adenoviral-mediated overexpression of HO-1 increased DAF expression, enhancing protection against C3 deposition and complement-mediated lysis, and this was reversed by DAF inhibitory monoclonal antibody (mAb) 1H4. Likewise, bilirubin, Fe chelation, and overexpression of heavy-chain ferritin all induced DAF expression in endothelial cells (EC). Analysis of cardiac endothelial cells isolated from Hmox1−/− mice revealed a 60% reduction in DAF expression compared with Hmox1+/+ EC, and Hmox1−/− cells showed enhanced sensitivity to complement. We propose that modulation of complement activation through induction of DAF represents an important component of the cytoprotective effects of HO-1 against vascular injury, such as that associated with posttransplant vasculopathy, allograft rejection, and ischemia reperfusion.

A Protein Kinase Cε-Anti-apoptotic Kinase Signaling Complex Protects Human Vascular Endothelial Cells against Apoptosis through Induction of Bcl-2

Endothelial cell apoptosis is associated with vascular injury and predisposes to atherogenesis. Endothelial cells express anti-apoptotic genes including Bcl-2, Bcl-XL and survivin, which also contribute to angiogenesis and vascular remodeling. We report a central role for protein kinase Cepsilon (PKCepsilon) in the regulation of Bcl-2 expression and cytoprotection of human vascular endothelium against apoptosis. Using myristoylated inhibitory peptides, a predominant role for PKCepsilon in vascular endothelial growth factor-mediated endothelial resistance to apoptosis was revealed. Immunoblotting of endothelial cells infected with an adenovirus expressing a constitutively active form of PKCepsilon (Adv-PKCepsilon-CA) or control Adv-beta-galactosidase demonstrated a 3-fold, PKCepsilon-dependent increase in Bcl-2 expression, with no significant change in Bcl-XL, Bad, Bak, or Bax. The induction of Bcl-2 inhibited apoptosis induced by serum starvation or etoposide, and PKCepsilon activation attenuated etoposide-induced caspase-3 cleavage. The functional role of Bcl-2 was confirmed with Bcl-2 antagonist HA-14-1. Inhibition of phosphoinositide 3-kinase attenuated vascular endothelial growth factor-induced protection against apoptosis, and this was rescued by overexpression of constitutively active PKCepsilon, suggesting PKCepsilon acts downstream of phosphoinositide 3-kinase. Co-immunoprecipitation studies demonstrated a physical interaction between PKCepsilon and Akt, which resulted in formation of a signaling complex, leading to optimal induction of Bcl-2. This study reveals a pivotal role for PKCepsilon in endothelial cell cytoprotection against apoptosis. We demonstrate that PKCepsilon forms a signaling complex and acts co-operatively with Akt to protect human vascular endothelial cells against apoptosis through induction of the anti-apoptotic protein Bcl-2 and inhibition of caspase-3 cleavage.

Statin-mediated cytoprotection of human vascular endothelial cells: a role for Kruppel-like factor 2-dependent induction of heme oxygenase-1.

Heme oxygenase-1 (HO-1), by exerting anti-inflammatory, antiproliferative, antiapoptotic and antioxidant effects in the vasculature, protects against atherosclerosis and post-transplant vasculopathy. We noted the overlap between the effects of HO-1 and those attributed to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins). This led to an investigation of the role of HO-1 in statin-mediated cytoprotection in primary human endothelial cells (ECs), and the ability of Kruppel-like factor 2 (KLF2) to regulate HO-1 function. Treatment of human umbilical vein and aortic ECs with atorvastatin significantly upregulated HO-1 promoter activity, mRNA expression and protein expression, increasing HO-1 enzymatic activity as shown by raised intracellular bilirubin IXalpha. This effect was indirect, dependent upon inhibition of HMG-CoA reductase and geranylgeranylation, and independent of nitric oxide or changes in mRNA stability. Atorvastatin protected ECs against the generation of reactive oxygen species and H(2)O(2)-induced injury. HO-1 inhibition, with small interfering RNA (siRNA) or zinc protoporphyrin IX, abrogated atorvastatin-mediated cytoprotection. Atorvastatin upregulated KLF2 expression, whereas KLF2 siRNA attenuated statin-induced HO-1 and its associated antioxidant cytoprotective effects. Iron chelation, adenoviral-mediated overexpression of ferritin or supplementation of culture media with biliverdin reversed the inhibitory effects of HO-1 and KLF2 siRNA, suggesting that bile pigments and ferritin mediate the antioxidant actions of statin-induced HO-1. We have identified a novel link between KLF2 and HO-1 in human vascular ECs, demonstrating that atorvastatin-mediated HO-1 upregulation, and its associated antioxidant effect, is KLF2-dependent. The relationship between KLF2 and HO-1 is likely to represent an important component of the vasculoprotective profile of statins.

Does Gender Influence the Likelihood or Types of Complications Following Cardiac Surgery?

Over 410,000 cardiac surgeries are performed in American women each year. Women having coronary artery bypass graft (CABG) and valve surgery do so at an older age and with more cardiovascular risk factors than men. Women's smaller body size may also increase risk by increasing the technical difficulty of surgical procedures. Female CABG patients appear to have higher perioperative mortality and cardiac morbidity, although studies of neurologic outcomes in female CABG patients have produced equivocal findings. Women undergoing CABG tend to consume more hospital resources than men do in terms of blood transfusion, mechanical ventilation, and length of intensive care unit and overall hospital stay. With regard to valve surgery, women appear to have worse outcomes than men if the surgery is combined with a CABG operation. Women and men undergoing isolated aortic valve surgery have similar mortality, but little is known about gender differences in mitral and tricuspid valve surgery outcomes. Women who require heart transplantation tend to have idiopathic cardiomyopathy rather than the ischemic cardiomyopathy that is more common in male heart transplant candidates. Although female heart transplant recipients seem to have a stronger immunologic response after transplantation, which manifests in more frequent acute rejection episodes, it is not clear whether this increases women's mortality risk. Men appear to have a greater incidence of posttransplant vasculopathy than women. Further research is needed to identify risk factors for perioperative morbidity and mortality in women undergoing cardiac surgery and to develop medical interventions to mitigate these risks.

HEART TRANSPLANT PATIENTS TREATED WITH SIROLIMUS IMMMUNOTHERAPY SHOW SIGNIFICANT REDUCTION IN THE PROGRESSION OF GRAFT VASCULOPATHY AT 2 YEARS

O408* Aims: Six-month results from a phase 2, open-label study of sirolimus-treated heart transplant patients demonstrated significant reductions in the incidence of acute rejection and the progression of transplant vasculopathy as assessed by intracoronary ultrasound (ICUS). The 2-year ICUS results are presented from this randomized trial. Methods: After heart transplantation, 136 patients were randomly assigned (2:1) to receive sirolimus or azathioprine in combination with cyclosporine and steroids. At 2 years, 58 patients (43%), 38 receiving sirolimus and 20 receiving azathioprine, underwent investigator-driven ICUS to determine if benefits on vasculopathy were sustained. Differences between groups were assessed using unpaired t test. Results: ICUS parameters were similar between groups at baseline. By 2 years, all parameters of transplant vasculopathy had progressed significantly in patients receiving azathioprine. Sirolimus-treated patients, however, showed significantly less intima and media proliferation as well as reduced lumen encroachment. ICUS results at 2 years are shown in the table.FigureICUS at 2 Years Conclusions: Sirolimus immunotherapy provides marked protection from transplant vasculopathy compared with azathioprine, not just at 6 months but also at 2 years after cardiac transplantation. These data represent the longest duration and greatest magnitude of effect of any agent on posttransplant vasculopathy.

THE RISK OF CORONARY TRANSPLANT VASCULOPATHY IN THE PATIENT BRIDGED WITH A VENTRICULAR ASSIST DEVICE

O343* Aims: A common consequence of left ventricular assist device (LVAD) implantation is the development of anti-HLA alloantibodies. While alloantibodies have been shown to be associated to vascular rejection the ultimate impact on vasculopathy is still ill defined. Previously observed the presence of Class I anti-HLA donor reactive antibodies was associated with significant early post-transplant vasculopathy. To examine this we obtained coronary angiograms from 196 patients bridged with LVAD and compared their post transplant coronary angiograms to a non-LVAD cohort (n=112) examined in a previous study. Methods: Adult patients undergoing orthotopic heart transplant between 1999-2000 were retrospectively studied and compared to our LVAD population. Coronary angiograms were retrospectively reviewed and severity of coronary vasculopathy was categorized as either normal, mild, moderate or severe. Other variables studied included cytotoxic panel reactive antibodies (PRA) against T-cell targets and flow cytometric crossmatching against donor t lymphocytes. Results: As anticipated there was an increase sensitization in those patients receiving a LVAD. Twenty-one percent of LVAD patients had a T-cell PRA greater than 10% at the time of transplant compared to 3.6% of the controls (p<0.0001, Fishers exact). Likewise, 25% of the LVAD patients had positive T-cell flow crossmatches compared to 5.4% of the controls (p<0.0001). Despite this coronary angiograms revealed no significant difference in the degree of coronary vasculopathy between the groups at follow-up. Normal coronary anatomy was detected in 72.5 % of LVAD patients and 64.6% of non-LVAD patients (p=0.79). These results were nearly identical at 2- and 3- year follow-up (71.8% vs. 64.6% and 58.3% vs.59.7%). Conclusions: While preoperative LVAD use is associated with a risk of recipient sensitization these patients develop transplant vasculopathy at the same rate as those not receiving a LVAD.

Presentation of MHC-disparate donor antigens predominantly by the indirect pathway results in the development of posttransplant vasculopathy: salutary effects of perioperative costimulatory blockade

Presentation of MHC-disparate donor antigens predominantly by the indirect pathway results in the development of posttransplant vasculopathy: salutary effects of perioperative costimulatory blockade

Endothelin-1 receptor blockade and its effect on chronic rejection

ENDOTHELIN-l (ET-l), a 21 amino acid peptide with potent vasoconstrictive properties has been implicated in the development of acute allograft rejection.) It is primarily produced by endothelial cells and its secretion, during acute rejection episodes, has been shown to be upregulated by TGFf3 and other proinflammatory cytokines released by graft infiltrating cells.) Additionally, ET-l has also been shown to facilitate proliferation of a-smooth muscle actin-positive (a-smA +) cells? Given that proliferation and/or accumulation of a-smA + cells is a prominent feature in neointimal thickening encountered in posttransplant vasculopathy, it is rational to propose that ET-l may play an important role in the pathogenesis of this lesion. To test this tenet, we proceeded to block ET-receptors (both ETA and ET B) by bosentan (a non peptide antagonist of ET -1) and to study its influence on the evolvement of chronic rejection (CR) in an established mouse model of aortic allotransplantation.3

Prevention of chronic rejection in mouse aortic allografts by combined treatment with CTLA4-Ig and anti-CD40 ligand monoclonal antibody.

In this study, using a murine model of aortic allotransplantation, the role of blockade of signaling through CD28/B7 and CD40/CD40 ligand costimulatory pathways in the evolvement of posttransplant vasculopathy was examined. Aortic allografts were transplanted across C57BL/1OJ (H2b)-->C3H (H2k) strain combinations. Transient or more stable blockade of second signaling was achieved by either a single injection or multiple injections of CTLA4-Ig fusion protein (200 microg/dose i.p.) and/or anti-CD40 ligand (CD40L) monoclonal antibody (250 microg i.m.). At day 30 after transplantation, the grafts were harvested for histopathological and immunohistochemical examination. Similar to allografts of untreated animals, aortic allografts obtained from recipients treated with either CTLA4-Ig or anti-CD40L monoclonal antibody alone exhibited marked narrowing of the lumen primarily due to concentric intimal thickening caused by proliferation of alpha-smooth muscle actin-positive cells. Contemporaneous treatment, however, with either a single injection or multiple injections of CTLA4-Ig and anti-CD40L monoclonal antibody resulted in marked diminution of intimal thickening. Interestingly, concurrent prolonged inhibition of CD28/B7 and CD40/CD40L pathways resulted in complete abrogation of the development of posttransplant arteriopathy. These data suggest that a more stable disruption of signaling through costimulatory pathways may be required to obviate the development of posttransplant vasculopathy.