Novel mediators of aneurysm progression in bicuspid aortic valve disease.

Bicuspid aortic valve (BAV) disease is a congenital abnormality that is associated with ascending aortic aneurysm yet many of the molecular mechanisms remain unknown. To identify novel molecular mechanisms of aneurysm formation we completed microarray analysis of the proximal (severely dilated) and distal (less dilated) regions of the ascending aorta from five patients with BAV. We identified 180 differentially expressed genes, 40 of which were validated by RT-qPCR. Most genes had roles in inflammation and endothelial cell function including cytokines and growth factors, cell surface receptors and the Activator Protein 1 (AP-1) transcription factor family (FOS, FOSB and JUN) which was chosen for further study. AP-1 was differentially expressed within paired BAV aneurysmal samples (n = 8) but not Marfan patients (n = 5). FOS protein was significantly enriched in BAV aortas compared to normal aortas but unexpectedly, ERK1/2 activity, an upstream regulator of FOS was reduced. ERK1/2 activity was restored when BAV smooth muscle cells were cultured in vitro. An mRNA-miRNA network within paired patient samples identified AP-1 as a central hub of miRNA regulation. FOS knockdown in BAV SMCs increased expression of miR-27a, a stretch responsive miRNA. AP-1 and miR-27a were also dysregulated in a mouse model of aortic constriction. In summary, this study identified a central role for AP-1 signaling in BAV aortic dilatation by using paired mRNA-miRNA patient sample. Upstream analysis of AP-1 regulation showed that the ERK1/2 signaling pathway is dysregulated and thus represents a novel chain of mediators of aortic dilatation in BAV which should be considered in future studies.

Increases in Serum Autoantibodies After Left Ventricular Assist Device Implantation.

BACKGROUND: Left ventricular assist devices (LVADs) can serve as a bridge to transplant or destination therapy for patients with advanced heart failure. Implantation of LVADs is known to be associated with increases in anti-HLA antibodies, but less is known about how autoantibody levels change with the use of these devices. METHODS AND RESULTS: Autoantibody levels were quantified with the use of customized antigen microarrays in 22 patients both before and after LVAD. We observed an increase (1.5- to 2-fold) in 14 IgG autoantibodies in the serum of patients after LVAD, including autoantibodies against cardiac proteins (myosin, troponin I, tropomyosin), DNA, and structural proteins (collagen, laminin). There was also a small but significant rise in total serum IgG after LVAD. Increases in autoantibodies after LVAD were positively associated with increases in calculated panel-reactive antibody class II (P = .05) and negatively correlated with age (r = -0.45; P < .05). Cytokines were evaluated to gain insights into the mechanism of antibody generation, and we observed a positive correlation between total IgG levels after LVAD and the level of monocyte chemoattractant protein 1 (r = 0.60; P < .05). CONCLUSIONS: LVAD implantation is associated with increases in IgG autoantibodies, anti-HLA antibodies, and total IgG. Increases in IgG after LVAD implantation may relate to an inflammatory response triggered by these devices.

Recipient hypertonic saline infusion prevents cardiac allograft dysfunction.

OBJECTIVE: Hypertonic saline (HTS) has potent immune and vascular effects. We assessed recipient pretreatment with HTS on allograft function in a porcine model of heart transplantation and hypothesized that HTS infusion would limit endothelial and left ventricular (LV) dysfunction following transplantation. METHODS: Heart transplants were performed after 6 hours of cold ischemic storage. Recipient pigs were randomized to treatment with or without HTS (7.5% NaCl) before cardiopulmonary bypass (CPB). Using a myograft apparatus, coronary artery endothelial-dependent (Edep) and -independent (Eind) relaxation was assessed. LV performance was determined using pressure-volume loop analysis. Pulmonary interleukin (IL)-2, IL-6, and tumor necrosis factor (TNF)-α expression was measured. RESULTS: Weaning from CPB and LV performance after transplantation were improved in HTS-treated animals. Successful weaning from CPB was greater in the HTS-treated hearts (8 of 8 vs 2 of 8; P < .05). Mean LV functional recovery was improved in the HTS-treated animals, as assessed by preload recruitable stroke work (65 ± 10% vs 27 ± 10%; P < .001) and end-systolic elastance (55 ± 7% vs 37 ± 4%; P < .001). Treatment with HTS resulted in improved Edep (mean maximum elastance [Emax], 56 ± 5% vs 37 ± 7%; P < .001) and Eind (mean Emax%, 77 ± 6% vs 52 ± 4%; P < .001) vasorelaxation compared with control. Pulmonary expression of IL-2, IL-6, and TNF-α increased following transplantation, whereas HTS therapy attenuated IL production (P < .001). Transplantation increased plasma TNF-α levels and LV TNF-α expression, whereas HTS prevented this up-regulation (P < .001). CONCLUSIONS: Recipient HTS pretreatment preserves allograft vasomotor and LV function, and HTS therapy limits CPB-induced injury. HTS may be a novel recipient intervention to prevent graft dysfunction.

Effects of everolimus and HLA-G on cellular proliferation and neutrophil adhesion in an in vitro model of cardiac allograft vasculopathy.

Human leukocyte antigen-G (HLA-G) expression is modulated by immunosuppressant use and is associated with lower incidence of graft rejection and cardiac allograft vasculopathy (CAV). We examined whether everolimus induces HLA-G expression and inhibits human coronary artery smooth muscle cell (HCASMC) proliferation, a critical event in CAV. Also, we examined whether TNFα-stimulated neutrophil adhesion is inhibited by HLA-G on human coronary artery endothelial cells (HCAECs). HLA-G expression in HCASMCs following everolimus treatment was determined by western-blot densitometric analysis. HCASMCs proliferation following incubation with recombinant HLA-G was determined by automated cell counter detecting 2-10 µm particles. Assessment of recombinant HLA-G on neutrophil adhesion to HCAECs in response to TNF-α induced-injury was determined by nonstatic adhesion assays. HLA-G expression was upregulated in HCASMCs following everolimus exposure (1000 ng/ml; P < .05). HLA-G (500, 1000 ng/ml; both P < .05) reduced HCASMC proliferation and inhibited TNFα-stimulated neutrophil adhesion to endothelial cells at all concentrations (0.1-1 ng/ml; all P < .001). Our study reveals novel regulation of HLA-G by everolimus, by demonstrating HLA-G upregulation and subsequent inhibition of HCASMC proliferation. HLA-G is a potent inhibitor of neutrophil adhesion to HCAECs. Findings support HLA-G's importance and potential use in heart transplantation for preventative therapy or as a marker to identify patients at high risk for developing CAV.

10-Year Experience with HLA-G in Heart Transplantation.

The Human Leukocyte Antigen-G (HLA-G) is a MHC-class Ib molecule with robust immunomodulatory properties; in transplant, it inhibits cytotoxic activity of immune cells and thus has a pivotal role in protecting the allograft from immune attack. The present review details a 10-year experience investigating the influence of HLA-G on heart transplantation, allograft rejection and cardiac allograft vasculopathy development. Exploration of HLA-G in transplantation began with the initial findings of its increased expression in allograft hearts. Since then, HLA-G has been recognized as an important factor in transplant immunology. We discuss inducers of HLA-G expression, and the importance of HLA-G as a potential biomarker in allograft rejection and heart failure. We also highlight the importance of polymorphisms and how they may influence both HLA-G expression and clinical outcomes. There remains much to be done in this field, however we hope that findings from our group and other groups will ignite interest and facilitate further expansion of HLA-G research in transplantation.

Progressive Aortic Dilation Is Regulated by miR-17-Associated miRNAs.

BACKGROUND: Patients with a bicuspid aortic valve (BAV) are at increased risk for progressive aortic dilation associated with extracellular matrix (ECM) degradation by matrix metalloproteinases (MMP). However, the mechanisms responsible for initiating this process are unknown. In the heart, MMP activity is regulated by micro-ribonucleic acid-17 (miR-17)-related downregulation of tissue inhibitors of metalloproteinases (TIMP); a similar process may exist in the aorta. OBJECTIVES: This study sought to ascertain whether aortic matrix degradation in BAV patients progresses by miR-17-related miRNA regulation of TIMP-MMP. METHODS: To eliminate confounding patient-related factors, severely dilated and less dilated aortic tissue samples were collected from 12 BAV patients. Gene and protein expression levels were evaluated in paired tissue samples from the same patient and were compared to aortic samples from 16 patients with aortas that appeared to be normal. RESULTS: Gene expression analyses confirmed increased expression of miR-17-related miRNAs in less dilated compared with severely dilated tissue from the same patient or normal aortic sample. TIMP-1, -2, and -3 were significantly decreased, and MMP2 activity was significantly increased in less dilated samples, suggesting that this normal-looking tissue was in the early stages of ECM degradation. Smooth muscle cells isolated from normal or BAV aortas transfected with an miR-17 mimic had decreased TIMP-1 and -2 expression and increased MMP2 activity, whereas the opposite effects were seen with an miR-17 inhibitor, suggesting that miR-17 may control the TIMP-MMP balance in these tissues. Luciferase reporter assays demonstrated that miR-17 regulated TIMP-1 and -2 expression. CONCLUSIONS: Our in vitro and in vivo studies taken together confirm that miR-17 directly regulates TIMP-1 and -2. Less dilated aortic BAV tissue may be in the initial stages of dilation under the control of miR-17-related miRNAs. New therapies that inhibit these miRNAs may prevent aortic dilation.

Generation of Antigen Microarrays to Screen for Autoantibodies in Heart Failure and Heart Transplantation.

Autoantibodies directed against endogenous proteins including contractile proteins and endothelial antigens are frequently detected in patients with heart failure and after heart transplantation. There is evidence that these autoantibodies contribute to cardiac dysfunction and correlate with clinical outcomes. Currently, autoantibodies are detected in patient sera using individual ELISA assays (one for each antigen). Thus, screening for many individual autoantibodies is laborious and consumes a large amount of patient sample. To better capture the broad-scale antibody reactivities that occur in heart failure and post-transplant, we developed a custom antigen microarray technique that can simultaneously measure IgM and IgG reactivities against 64 unique antigens using just five microliters of patient serum. We first demonstrated that our antigen microarray technique displayed enhanced sensitivity to detect autoantibodies compared to the traditional ELISA method. We then piloted this technique using two sets of samples that were obtained at our institution. In the first retrospective study, we profiled pre-transplant sera from 24 heart failure patients who subsequently received heart transplants. We identified 8 antibody reactivities that were higher in patients who developed cellular rejection (2 or more episodes of grade 2R rejection in first year after transplant as defined by revised criteria from the International Society for Heart and Lung Transplantation) compared with those who did have not have rejection episodes. In a second retrospective study with 31 patients, we identified 7 IgM reactivities that were higher in heart transplant recipients who developed antibody-mediated rejection (AMR) compared with control recipients, and in time course studies, these reactivities appeared prior to overt graft dysfunction. In conclusion, we demonstrated that the autoantibody microarray technique outperforms traditional ELISAs as it uses less patient sample, has increased sensitivity, and can detect autoantibodies in a multiplex fashion. Furthermore, our results suggest that this autoantibody array technology may help to identify patients at risk of rejection following heart transplantation and identify heart transplant recipients with AMR.

Increased cyclic guanosine monophosphate levels and continuous-flow left-ventricular assist devices: Implications for gastrointestinal bleeding.

OBJECTIVES: We examine the hypothesis that cyclic guanosine monophosphate (cGMP) levels are elevated in recipients of continuous-flow left ventricular assist devices (CF-LVADs) and that elevated cGMP levels are associated with a risk of gastrointestinal (GI) bleeding events. METHODS: The levels of cGMP, nitric oxide, platelet activation markers, platelet-derived growth factors (PDGF) AB/BB and AA, and the inflammatory mediator C-reactive protein (CRP) were examined in 19 CF-LVAD recipients, 21 patients who had heart failure, and 19 healthy control-group participants. RESULTS: The median level of cGMP was significantly higher in CF-LVAD recipients, compared with healthy participants (6.6 vs 2.1 pmol/mL, u = 62.5; P = .001; r = -0.55). Median cGMP levels in the heart failure group (12.5 pmol/L) were higher, compared with both CF-LVAD recipients (u = 75.0; P = .001; r = -0.53) and healthy participants (u = 4.0; P < .001; r = -0.83). Compared with the healthy group, median CRP levels were significantly higher in CF-LVAD recipients (2.9 vs 8.0 mg/L; u = 58.0; P < .001; r = -0.63) and heart failure patients (2.9 vs 7.0 mg/L; u = 59.0; P < .001; r = -0.65). In the subgroup of patients supported with the HeartMate II (Thoratec Corporation, Pleasanton, Calif), pulsatility index was significantly negatively correlated with cGMP levels (r = -0.73; P < .05), indicating that low pulsatility index is associated with higher cGMP levels. High cGMP levels were significantly associated with GI bleeding events, but not with bleeding events in general. CONCLUSIONS: The primary finding of this study is that GI bleeding in CF-LVAD recipients is associated with significantly elevated cGMP levels, despite high levels of CRP, which interfere with cGMP production. Further studies are required to determine whether elevated cGMP levels can be used as a clinical marker for increased risk of GI bleeding in CF-LVAD recipients.

New Developments in HLA-G in Cardiac Transplantation.

Human Leukocyte Antigen-G (HLA-G) is a non-classical class 1b protein, whose gene is located on chromosome 6 (6p21.31). HLA-G inhibits the immune cells' cytotoxic activity by interacting with specific receptors on their membranes. Since it is a naturally occurring immune modulator, HLA-G has been investigated in transplantation. Indeed, a number of investigations reveal that HLA-G expression is influenced by genetic polymorphisms and in turn, those polymorphisms are associated with detrimental or beneficial outcomes in various pathological situations. The present review introduces the HLA-G molecule, the gene and its polymorphisms. It focuses on the expression of HLA-G and the role of polymorphisms primarily in heart transplant outcomes, secondarily in other transplant organs, as well as the role of the allograft and effect of medical therapy. We discuss the limitations in HLA-G transplant investigations and future directions. The immune inhibiting activity of HLA-G has a great deal of potential for its utilization in enhancing diagnostic, preventive and therapeutic strategies against rejection in the setting of transplantation.

The effect of insulin to decrease neointimal growth after arterial injury is endothelial nitric oxide synthase-dependent.

In vitro, insulin has mitogenic effects on vascular smooth muscle cells (VSMC) but also has protective effects on endothelial cells by stimulating nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) expression. Furthermore, NOS inhibition attenuates the effect of insulin to inhibit VSMC migration in vitro. Using an in vivo model, we have previously shown that insulin decreases neointimal growth and cell migration and increases re-endothelialization after arterial injury in normal rats. Since insulin can stimulate NOS, and NO can decrease neointimal growth, we hypothesized that NOS, and more specifically eNOS was required for the effects of insulin in vivo. Rats were given subcutaneous insulin implants (3 U/day) alone or with the NOS inhibitor l-NAME (2 mg kg(-1) day(-1)) 3 days before arterial (carotid or aortic) balloon catheter injury. Insulin decreased both neointimal area (P < 0.01) and cell migration (P < 0.01), and increased re-endothelialization (P < 0.05). All of these effects were prevented by the co-administration of l-NAME. Insulin was found to decrease inducible NOS expression (P < 0.05) but increase eNOS phosphorylation (P < 0.05). These changes were also translated at the functional level where insulin improved endothelial-dependent vasorelaxation. To further study the NOS isoform involved in insulin action, s.c. insulin (0.1 U/day) was given to wild-type and eNOS knockout mice. We found that insulin was effective at decreasing neointimal formation in wild-type mice after wire injury of the femoral artery, whereas this effect of insulin was absent in eNOS knockout mice. These results show that the vasculoprotective effect of insulin after arterial injury is mediated by an eNOS-dependent mechanism.

Longitudinal assessment of inflammation in recipients of continuous-flow left ventricular assist devices.

BACKGROUND: The long-term effects of continuous-flow left ventricular assist device (CF-LVAD) support on trends of inflammatory markers over time are unknown. We examined the hypothesis that the levels of inflammatory markers in CF-LVAD recipients are higher than in healthy controls and that these levels increase over time with long-term CF-LVAD support. METHODS: We examined the levels of inflammatory markers longitudinally at baseline before CF-LVAD implantation and at 3, 6, and 9 months after implantation. We then compared the levels of inflammatory markers to those in a healthy control group. RESULTS: Compared with baseline values before CF-LVAD implantation, left ventricular end-diastolic diameter (LVEDd) and left ventricular end-systolic diameter (LVESd) decreased significantly at 3, 6, and 9 months after CF-LVAD implantation. Brain natriuretic peptide (BNP) levels dropped significantly after CF-LVAD implantation but did not normalize. Improvements in ejection fraction at 3, 6, and 9 months after CF-LVAD implantation did not reach significance. Monocyte chemoattractant protein-1, interferon γ-induced protein, and C-reactive protein levels were higher in the CF-LVAD recipients at each of the time points (baseline before CF-LVAD implantation and 3, 6, and 9 months after implantation) compared with levels in healthy controls. In CF-LVAD recipients, serum interleukin-8, tumour necrosis factor-α, and macrophage inflammatory protein-ß increased significantly at 9 months, and macrophage-derived chemokine increased at 6 months after CF-LVAD implantation compared with baseline. CONCLUSIONS: Despite improvements in LV dimensions and BNP levels, markers of inflammation remained higher in CF-LVAD recipients. High levels of inflammation in CF-LVAD recipients may result from heart failure preconditioning or the long-term device support, or both. Because inflammation may be detrimental to CF-LVAD recipients, future studies should determine whether inflammatory pathways are reversible.

Markers of inflammation in recipients of continuous-flow left ventricular assist devices.

Although the newer continuous-flow left ventricular assist devices (CF-LVADs) provide clinical advantages over the pulsatile pumps, the effects of low pulsatility on inflammation are incompletely understood. The objective of our study was to examine the levels of inflammatory mediators in CF-LVAD recipients compared with both healthy control subjects and heart failure patients who were candidates for CF-LVAD support. Plasma levels of chemokines, cytokines, and inflammatory markers were measured in 18 CF-LVAD recipients and compared with those of 14 healthy control subjects and 14 heart failure patients who were candidates for CF-LVADs. The levels of granulocyte macrophage-colony stimulating factor, macrophage inflammatory proteins-1ß, and macrophage-derived chemokine were significantly higher in the CF-LVAD group compared with both the heart failure and the healthy control groups, whereas no significant differences were observed between the healthy control subjects and the heart failure groups. Compared with the healthy controls, C-reactive protein, interferon gamma-induced protein-10, monocyte chemotactic protein-1, and interleukin-8 levels were significantly higher in both the CF-LVAD and heart failure groups, but no significant differences were observed between the CF-LVAD recipients and the heart failure patients. Inflammatory markers were elevated in CF-LVAD recipients compared with healthy control subjects and the heart failure patients. Further studies should investigate the clinical implications of elevated levels of inflammation in CF-LVAD recipients.

Human leukocyte antigen-G is upregulated in heart failure patients: a potential novel biomarker.

Immune activation and inflammation play critical roles in the development of heart failure (HF). Human leukocyte antigen-G (HLA-G) is a nonclassical, major histocompatibility complex class I (MHC-I) protein, upregulated in the context of transplantation, malignancy, and inflammation, and has been correlated with various clinical outcomes. We sought to evaluate the utility of plasma HLA-G in identifying patients with HF. We conducted a single-center, cross-sectional pilot study involving 82 patients diagnosed with HF and 10 healthy controls. Concentrations of circulating HLA-G and inflammatory markers were detected with specific enzyme-linked immunosorbent assay kits and quantified according to purified protein standards. The mean age of the patients was 49.1 ± 12.0 years and 62.2% were male. The median and interquartile range of HLA-G levels (U/ml) were significantly higher (p < 0.001) in HF patients (63, 36-98) compared with controls (28, 22-40). Moreover, HLA-G levels that were similarly (p = 0.766) upregulated across all New York Heart Association functional classes. There was no significant correlation between serum HLA-G and other biomarkers. In conclusion, HLA-G is upregulated in patients diagnosed with HF. Its marked elevation even in New York Heart Association class I patients might indicate that serum HLA-G is a more sensitive marker than other classical HF biomarkers.

Epidermal growth factor-like domain 7 is a novel inhibitor of neutrophil adhesion to coronary artery endothelial cells injured by calcineurin inhibition.

BACKGROUND: We investigated the effect of epidermal growth factor-like domain 7 (Egfl7) on nuclear factor-κB activation, intercellular adhesion molecule-1 expression, and neutrophil adhesion to human coronary artery endothelial cells after calcineurin-inhibition-induced injury. METHODS AND RESULTS: Human coronary endothelial cells were incubated with cyclosporine (CyA) 10 µg/mL with or without Egfl7 (100 ng/mL) or the Notch receptor activator Jagged1 (200 ng/mL) for 6 to 48 hours. CyA upregulated nuclear factor-κB (p65) activity (128 ± 2% of control, P<0.001) in nuclear extracts, as determined with a DNA-binding activity ELISA. This activity was inhibited by Egfl7 (86 ± 3% of control; P<0.001 versus CyA alone). Jagged1 blocked Egfl7-induced nuclear factor-κB inhibition (105 ± 4% of control; P<0.05 versus CyA plus Egfl7). CyA upregulated cell-surface intercellular adhesion molecule-1 expression (215 ± 13% of control; P<0.001), as determined by flow cytometry. This expression was suppressed by Egfl7 (148 ± 5%; P<0.001 versus CyA alone). Jagged1 attenuated the intercellular adhesion molecule-1-suppressive effect of Egfl7 when administered with CyA (193 ± 3% versus 148 ± 5%; P<0.01). CyA increased neutrophil adhesion to human coronary endothelial cells (control 20 ± 5%, CyA 37 ± 3%; P<0.001 versus control) in a nonstatic neutrophil adhesion assay. This increase was attenuated by Egfl7 (22 ± 6%; P<0.001 versus CyA alone). Jagged 1 attenuated the effect of Egfl7 on neutrophil adhesion (31±3%; P<0.001 versus Egfl7 plus CyA). CONCLUSIONS: Our study reveals that Egfl7 is a potent inhibitor of neutrophil adhesion to human coronary endothelial cells subsequent to calcineurin-inhibition-induced injury. Mechanistically, Egfl7 blocked nuclear factor-κB pathway activation and intercellular adhesion molecule-1 expression, which suggests that it may have significant antiinflammatory properties. Because Jagged1 blocked the effect of Egfl7, Notch receptor antagonism may contribute to the mechanism of action of Egfl7.

Tacrolimus preserves vasomotor function and maintains vascular homeostasis.

BACKGROUND: Post-transplant immunosuppression is associated with endothelial dysfunction that may lead to vasculopathy. We have previously demonstrated that cyclosporine causes vascular dysfunction. In this we study examined the effect of tacrolimus (Tac) in an identical model. METHODS: Lewis rats (n = 8 per group) were injected with Tac (low, medium or high dose) or saline (Con) daily for 2 weeks. Segments of thoracic aorta (TAo) were assessed for endothelium-dependent (Edep) and -independent (Eind) vasorelaxation (E(max)) and sensitivity to endothelin (ET)-induced vasoconstriction (C(max)). ET(A) and ET(B) receptor (Rc) expression levels were determined in TAo. Tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-ß) expression were determined in myocardial (LV) tissue. Plasma ET levels and tissue oxidative injury were quantified by enzyme-linked immunoassay. RESULTS: Tac did not impair Edep relaxation when compared with Con (p = 0.69). Impairment of sodium nitroprusside-mediated Eind vasorelaxation was noted with Tac (E(max): Con 69 ± 2%, Tac high 54 ± 2%; p = 0.0001), whereas no such impairment was seen with diltiazem-mediated Eind vasorelaxation (p = 0.06). Tac decreased sensitivity to ET (C(max): Con 222 ± 19%, Tac high 162 ± 11%; p = 0.0002) and ET levels (Con 0.8 ± 0.1 fmol/ml, Tac 0.4 ± 0.1 fmol/ml; p = 0.02). Tac did not alter ET(A) Rc expression (p = 0.28), but increased ET(B) Rc levels (p = 0.02). Oxidative injury was similar in both LV (p = 0.43) and Ao (p = 0.73) tissue. Similarly, TNF-α expression (p = 0.16) was not different between groups, whereas expression of TGF-ß demonstrated a significant decrease with Tac treatment (p = 0.02). CONCLUSION: Our findings suggests that tacrolimus has beneficial effects with respect to endothelial function.

Endothelin-1 antagonism and nitric oxide augmentation prevents cyclosporine-induced vasomotor impairment.

BACKGROUND: We previously demonstrated that cyclosporine (CyA) impairs endothelial function as a result of alterations in nitric oxide (NO) and endothelin-1 (ET-1) regulation. Bosentan (BOS), an ET-1 antagonist, and tetrahydrobiopterin (BH4), an eNOS cofactor, may reduce endothelial dysfunction by improving ET-1/NO homeostasis. METHODS: Lewis rats received intraperitoneal injections of CyA with BOS or with BOS+BH4 daily for 2 weeks. Control (Con) animals received saline injections. Thoracic aortic segments were assessed for endothelial-dependent (E(dep)) and -independent (E(ind)) relaxation (E(max%)) after exposure to acetylcholine and sodium nitroprusside. Vessel sensitivity to ET-1-induced vasospasm was evaluated. RESULTS: CyA use resulted in impaired E(dep) vasorelaxation when compared with Con, whereas BOS and BH4 treatment preserved E(dep) vasorelaxation. CyA significantly altered E(ind) vasorelaxation, whereas BOS and BH4 therapy attenuated CyA-induced effects. Compared with Con, CyA and BH4 exposure demonstrated increased sensitivity to ET-1 vasospasm. BOS therapy abrogated the CyA and BH4-induced sensitivity to vasospasm. CyA treatment resulted in higher 8-isoprostane levels compared with Con. CyA-mediated vascular dysfunction is characterized by impaired NO and ET-1 homeostasis. CONCLUSIONS: Our study suggests potential therapeutic strategies to prevent endothelial dysfunction as combined therapy with ET-1 antagonism and NO augmentation completely abrogated CyA-induced vascular injury.

Epidermal growth factor-like domain 7 suppresses intercellular adhesion molecule 1 expression in response to hypoxia/reoxygenation injury in human coronary artery endothelial cells.

BACKGROUND: Epidermal growth factor-like domain 7 (Egfl7) is a chemoattractant for endothelial cells, and its expression is restricted to endothelial cells. Hypoxia/reoxygenation (H/R) induced endothelial injury that occurs during transplantation contributes to the subsequent development of allograft vasculopathy. We investigated the effect of Egfl7 on endothelial cell intercellular adhesion molecule 1 expression in response to H/R injury. METHODS AND RESULTS: Human coronary artery endothelial cells were submitted to hypoxia (0.1% O(2)) followed by normoxia (21% O(2)) in the presence or absence of Egfl7 (100 ng/mL). Hypoxia alone increased the expression of Egfl7×140±8% of control at 3 hours (n=6; P<0.05) and 385±50% of control at 6 hours (n=6; P<0.001). Incubation with Egfl7 during the reoxygenation period prevented intercellular adhesion molecule 1 upregulation (mean fluorescence intensity: 5.37±0.92 versus 3.81±0.21; P<0.05; n=4 per group). Nuclear factor-κB nuclear localization on H/R injury was blocked by Egfl7 administration (cytosolic/nuclear ratio of 0.93±0.01 versus 1.44±0.24; P<0.05; n=4 per group). Inhibitor of nuclear factor-κB protein level was significantly reduced on H/R injury (26±4.6% of control expression; P<0.05; n=4 per group); however, concurrent incubation with Egfl7 attenuated this reduction (46±6.2% of control expression; P<0.05 when compared with H/R injury alone; n=4 per group). CONCLUSIONS: Our study reveals the novel observation that hypoxia upregulates human coronary artery endothelial cells expression of Egfl7 and that Egfl7 inhibits expression of intercellular adhesion molecule 1 subsequent to H/R injury. Mechanistically, Egfl7 prevented nuclear factor-κB nuclear localization and augmented inhibitor of nuclear factor-κB protein levels, suggesting that it inhibits nuclear factor-κB activation, a key step in the inflammatory activation of endothelial cells. Egfl7 may be protective against H/R injury incurred during transplantation and may modulate the events that lead to the development of graft vasculopathy.

Correlation between circulating endothelial progenitor cell function and allograft rejection in heart transplant patients.

Endothelial progenitor cells (EPCs) may contribute to rejection and cardiac allograft vasculopathy (CAV) by being intrinsically involved in the rejection process and causing neointimal hyperplasia. The mammalian target of rapamycin inhibitors (mTORi), sirolimus and everolimus, have been demonstrated to attenuate the progression of CAV and are cytotoxic to EPC. Thus, one mechanism by which mTORi may protect against CAV is by altering EPC function. Our study measured circulating EPC function and correlated this assessment with rejection episodes in heart transplant (HT) recipients. In addition, we examined the effect of mTORi on EPCs. Patients who received HT at our institution between 1995 and 2007 were included and stratified by International Society for Heart and Lung Transplantation (ISHLT) rejection grade. Group A (n = 13) consisted of patients with at least one moderate/severe rejection episode (grade > or = 2). Group B (n = 28) patients had no moderate/severe episodes (grade < 2). Patients were also independently stratified based on exposure as mTORi (n = 21) vs. non mTORi (n = 20). To assess EPC functional capacity, we counted the number of colony-forming units (CFU) of EPCs in peripheral blood samples from HT recipients. There were no significant differences in baseline characteristics between groups. The mean EPC-CFU counts/plate for group A (rejecting) were 30 +/- 6 vs.16 +/- 3 for group B (nonrejecting) (P = 0.03). The EPC-CFU counts/plate in the mTORi group (15 +/- 3) were lower compared to the non mTORi (27 +/- 5) group (P = 0.04). We found that EPC colony-forming capacity was higher in HT patients who experienced moderate/severe rejection episodes. Patients on mTORi showed a reduced EPC colony count consistent with our previous findings of EPC cytotoxicity. Detection of circulating EPC function post-transplant may reliably identify patient risk level for subsequent allograft rejection and allow for appropriate adjustments to immunosuppression. Converting to mTORi therapy may reduce EPC function and provide a novel mechanism to prevent rejection and possibly attenuate the development of CAV.

Donor pretreatment with hypertonic saline attenuates primary allograft dysfunction: a pilot study in a porcine model.

BACKGROUND: Hypertonic saline (HTS) has been previously demonstrated to have immune modulatory and vascular protective effects. We assessed the effect of donor pretreatment with HTS on allograft preservation in a porcine model of orthotopic heart transplantation. METHODS AND RESULTS: Orthotopic transplants were performed after 6 hours of cold static allograft storage. Donor pigs were randomly assigned to pretreatment with (n=7) or without (n=6) HTS (4.5 mL/kg of 7.5% NaCl) administered 1 hour before donor heart arrest. Administration of HTS increased serum sodium level from 138+/-2 mmol/L to 154+/-4 mmol/L, which normalized to 144+/-3 mmol/L 1 hour after infusion. Successful weaning from cardiopulmonary bypass was significantly greater in HTS-treated hearts (6/7 vs 1/6; P=0.029). Preload recruitable stroke work after transplantation was improved compared to control (88+/-21% vs 35+/-8% of baseline; P=0.0001). Similarly, end-systolic elastance was improved compared to control (85+/-17% vs 42+/-12% of baseline; P=0.0002). Posttransplantation systolic blood pressure was significantly higher in the donor HTS group (60+/-9 mm Hg vs 35+/-6 mm Hg; P=0.04). Donor HTS treatment improved coronary artery endothelial-dependent vasorelaxation compared with control (Emax: HTS, 59+/-4%; control, 47+/-3%; P=0.04). HTS also resulted in improved endothelial-independent vasorelaxation compared with control (Emax: HTS, 71+/-3%; control, 59+/-4%; P=0.03; ED-50: HTS, 0.56x10 to 6+/-0.23 mol/L; control, 2.5x10 to 6+/-1.0 mol/L; P=0.04). Sensitivity to endothelin-1-induced vasospasm was reduced with HTS pretreatment (% maximum contraction [Cmax]: HTS, 338+/-15%; control, 419+/-40%; P=0.01). CONCLUSIONS: Donor HTS pretreatment attenuates posttransplantation cardiac allograft myocardial dysfunction, improves posttransplantation systemic hemodynamic function, and preserves posttransplantation cardiac allograft vascular function. HTS may be a novel organ donor intervention to prevent primary graft dysfunction.