Shared genetic pathways contribute to risk of hypertrophic and dilated cardiomyopathies with opposite directions of effect.

The heart muscle diseases hypertrophic (HCM) and dilated (DCM) cardiomyopathies are leading causes of sudden death and heart failure in young, otherwise healthy, individuals. We conducted genome-wide association studies and multi-trait analyses in HCM (1,733 cases), DCM (5,521 cases) and nine left ventricular (LV) traits (19,260 UK Biobank participants with structurally normal hearts). We identified 16 loci associated with HCM, 13 with DCM and 23 with LV traits. We show strong genetic correlations between LV traits and cardiomyopathies, with opposing effects in HCM and DCM. Two-sample Mendelian randomization supports a causal association linking increased LV contractility with HCM risk. A polygenic risk score explains a significant portion of phenotypic variability in carriers of HCM-causing rare variants. Our findings thus provide evidence that polygenic risk score may account for variability in Mendelian diseases. More broadly, we provide insights into how genetic pathways may lead to distinct disorders through opposing genetic effects.

Human genetic determinants of the gut microbiome and their associations with health and disease: a phenome-wide association study.

Small-scale studies have suggested a link between the human gut microbiome and highly prevalent diseases. However, the extent to which the human gut microbiome can be considered a determinant of disease and healthy aging remains unknown. We aimed to determine the spectrum of diseases that are linked to the human gut microbiome through the utilization of its genetic determinants as a proxy for its composition. 180 single nucleotide polymorphisms (SNPs) known to influence the human gut microbiome were used to assess the association with health and disease outcomes in 422,417 UK Biobank participants. Potential causal estimates were obtained using a Mendelian randomization (MR) approach. From the total sample analysed (mean age was 57 ± 8 years), 194,567 (46%) subjects were male. Median exposure was 66-person years (interquartile range 59-72). Eleven SNPs were significantly associated with 28 outcomes (Bonferroni corrected P value < 4.63·10-6) including food intake, hypertension, atopy, COPD, BMI, and lipids. Multiple SNP MR pointed to a possible causal link between Ruminococcus flavefaciens and hypertension, and Clostridium and platelet count. Microbiota and their metabolites might be of importance in the interplay between overlapping pathophysiological processes, although challenges remain in establishing causal relationships.

Genetically Determined ABO Blood Group and its Associations With Health and Disease.

OBJECTIVE: To determine the spectrum of phenotypes linked to the ABO blood group system, using genetic determinants of the ABO blood group system. Approach and Results: We assessed the risk of 41 health and disease outcomes, and 36 linear traits associated with the ABO blood group system in the UK Biobank cohort. A total of 406 755 unrelated individuals were included in this study. Blood groups A, B, and O were determined based on allele combinations of previously established single-nucleotide polymorphisms rs8176746, rs8176719 in the ABO gene. Group AB was excluded because of its relative small sample size. Overall, 187 387 (46%) were male with a mean (SD) age of 57±8.1 years and a median total exposure of 64 person-years (interquartile range, 57-70). Of 406 755 individuals, 182 621 (44.9%) participants had blood group O, 182 786 (44.9%) had blood group A, and 41 348 (10.2%) had blood group B. ABO blood groups were associated with 11 health and disease outcomes (P<2.19×10-4). ABO blood groups were primarily associated with cardiovascular outcomes. Compared with individuals with blood group O, blood groups A and B were associated with increased odds of up to 1.56 (95% CI, 1.43-1.69) for thromboembolic events and decreased odds for hypertension (0.94 [95% CI, 0.92-0.97]). CONCLUSIONS: The ABO blood group system is associated with several parameters of healthy aging and disease development. Knowledge of ABO blood groups might be of interest for more personalized approaches towards health maintenance and the prevention of diseases.

Leukocyte profiles across the cardiovascular disease continuum: A population-based cohort study.

INTRODUCTION: Inflammation plays a pivotal role across all stadia of the cardiovascular disease (CVD) continuum, i.e. non-obstructive coronary artery disease (CAD), myocardial infarction (MI), and ischemic heart failure (iHF). However, inflammation across CVD continuum has not been studied yet within one population. Therefore, we mapped leukocyte profiles across the continuum within the UK Biobank. METHODS: The UK Biobank cohort study includes >500,000 participants aged 40 to 70 years who were recruited from 22 assessment centers across the United Kingdom from 2006 to 2010. A total of 333,218 individuals with available laboratory measurements at baseline were included in this study. These consisted of controls and individuals who had progression of CVD during follow-up (i.e. who developed CAD, MI, or iHF during follow-up). We investigated whether leukocytes and subtypes of leukocytes at baseline differed among the CVD continuum. Furthermore, we studied the possible interactions between sex and CVD on leukocytes. RESULTS: Of 333,218 individuals, 325,054 (97.5%) individuals were categorized as controls, and 8164 (2.5%) individuals had progression of CVD during follow-up. Of those 8164 individuals, 4552 (1.4%) developed CAD during follow-up, 2839 (0.9%) MI, and 773 (0.2%) in iHF. Compared to controls, mean leukocyte levels at baseline increased across the CVD continuum from 6.8·109 cells/L (SD 1.7·109 cells/L) to 7.7·109 cells/L (SD 1.9·109 cells/L) (Ptrend = 2.19·10-132) in individuals who developed iHF. This increase mainly depended on an increase in neutrophils. Furthermore, controls with leukocyte levels in the highest quartile at baseline had a 1.44 higher chance of being diagnosed with CAD during follow-up compared with individuals with leukocyte levels in lower quartiles (OR 1.44, 95% CI 1.34-1.56 P = 9.63·10-21). A similar increased change was observed for neutrophils, lymphocytes, monocytes, and eosinophils. There was a significant interaction between sex and CVD continuum on lymphocytes (P = 8.49·10-5). CONCLUSION: Overall leukocyte count increased across the CVD continuum, which mainly depended on the increase in neutrophil count. High leukocytes in individuals not having CAD at baseline were predictive for the development of CAD during follow-up. Women had a greater increase of lymphocytes across the CVD continuum compared to men. Understanding which cells are key players in which stadium, could serve as a starting point for the identification of new potential therapeutic targets in CVD.

Plasma interleukin 6 levels are associated with cardiac function after ST-elevation myocardial infarction.

BACKGROUND AND AIMS: Myocardial infarction triggers an inflammatory response involved in cardiac repair. We studied the association of the interleukin 6 (IL-6) cascade with infarct size and cardiac function after ST-elevation myocardial infarction (STEMI). METHODS: In 369 STEMI patients IL-6, soluble IL-6 receptor (sIL-6R), and soluble glycoprotein (sgp) 130 were measured at baseline (hospital admission), 24 h, 2 weeks, 7 weeks, 4 months, and 1 year post-PCI and sIL-6R/IL-6 ratio was calculated. At 4 months, infarct size and left ventricular ejection fraction (LVEF) were assessed by magnetic resonance imaging. Diastolic function (E/e') was determined by echocardiography. RESULTS: Hospital admission levels for IL-6, sIL-6R, sgp 130 were 3.7 pg/ml (IQR 2.1-6.7 pg/ml), 51.6 ng/ml (IQR 37.3-69.0 ng/ml), and 332 ng/ml (IQR 280-399 ng/ml), respectively. 24 h after admission, IL-6 had increased threefold compared to baseline (p < 0.001) and returned below baseline (p < 0.001) 2 weeks after STEMI. sIL-6R and sgp130 levels at 24 h remained similar to baseline but were increased at 2 weeks (p < 0.001; p < 0.001, respectively). IL-6 and sIL-6R/IL-6 ratio at 24 h were independently associated with infarct size [ß 5.4 (95% CI 3.3-7.5); p < 0.001, ß - 4.0 (95% CI - 6.1 to - 1.9); p < 0.001, respectively]. Higher levels of IL-6 at 24 h were associated with lower LVEF [ß - 4.2 (95% CI -6.7 to - 1.8); p = 0.001]. CONCLUSIONS: Higher IL-6 and lower sIL-6R/IL-6 ratio early after presentation with STEMI are indicative for larger infarct size and decreased cardiac function at 4 months.

Translational overview of cytokine inhibition in acute myocardial infarction and chronic heart failure.

Many cytokines are currently under investigation as potential target to improve cardiac function and outcome in the setting of acute myocardial infarction (MI) or chronic heart failure (HF). Here we aim to provide a translational overview of cytokine inhibiting therapies tested in experimental models and clinical studies. In various experimental studies, inhibition of interleukin-1 (IL-1), -6 (IL-6), -8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), CC- and CXC chemokines, and tumor necrosis factor-α (TNF-α) had beneficial effects on cardiac function and outcome. On the other hand, neutral or even detrimental results have been reported for some (IL-1, IL-6, IL-8, and MCP-1). Ambivalence of cytokine function, differences in study designs, treatment regimens and chosen endpoints hamper the translation of experimental research into clinical practice. Human studies are currently limited to IL-1ß inhibition, IL-1 receptor antagonists (IL-1RA), IL-6 receptor antagonists (IL-6RA) or TNF inhibition. Despite favorable effects on cardiovascular events observed in retrospective cohort studies of rheumatoid arthritis patients treated with TNF inhibition or IL-1RA, most prospective studies reported disappointing and inconsistent results. Smaller studies (n < 100) generally reported favorable results of anticytokine therapy on cardiac function, but only one of the larger studies (n > 100) evaluating IL-1ß inhibition presented positive results on outcome. In conclusion, of the 10 anticytokine therapies tested in animals models beneficial effects have been reported in at least one setting. In larger clinical studies, findings were unsatisfactory in all but one. Many anticytokine therapies with promising animal experimental data continue to require further evaluation in humans.

The epigenetic factor PCAF regulates vascular inflammation and is essential for intimal hyperplasia development.

OBJECTIVE: Genetic P300/CBP-associated factor (PCAF) variation affects restenosis-risk in patients. PCAF has lysine acetyltransferase activity and promotes nuclear factor kappa-beta (NFκB)-mediated inflammation, which drives post-interventional intimal hyperplasia development. We studied the contributing role of PCAF in post-interventional intimal hyperplasia. METHODS AND RESULTS: PCAF contribution to inflammation and intimal hyperplasia was assessed in leukocytes, macrophages and vascular smooth muscle cells (vSMCs) in vitro and in a mouse model for intimal hyperplasia, in which a cuff is placed around the femoral artery. PCAF deficiency downregulate CCL2, IL-6 and TNF-alpha expression, as demonstrated on cultured vSMCs, leukocytes and macrophages. PCAF KO mice showed a 71.8% reduction of vSMC-rich intimal hyperplasia, a 73.4% reduction of intima/media ratio and a 63.7% reduction of luminal stenosis after femoral artery cuff placement compared to wild type (WT) mice. The association of PCAF and vascular inflammation was further investigated using the potent natural PCAF inhibitor garcinol. Garcinol treatment reduced CCL2 and TNF-alpha expression, as demonstrated on cultured vSMCs and leukocytes. To assess the effect of garcinol treatment on vascular inflammation we used hypercholesterolemic ApoE*3-Leiden mice. After cuff placement, garcinol treatment resulted in reduced arterial leukocyte and macrophage adherence and infiltration after three days compared to untreated animals. CONCLUSIONS: These results identify a vital role for the lysine acetyltransferase PCAF in the regulation of local inflammation after arterial injury and likely the subsequent vSMC proliferation, responsible for intimal hyperplasia.

High-sensitivity C-reactive protein and long term reperfusion success of primary percutaneous intervention in ST-elevation myocardial infarction.

AIMS: In STEMI patients, success of reperfusion of primary PCI predicts cardiac remodeling and clinical outcome. This success may depend on inflammation. We aimed to investigate the association between inflammation and reperfusion success, left ventricular function and long-term mortality in STEMI patients. METHODS: In 376 consecutive STEMI patients of the GIPS-III trial hs-CRP levels were measured at baseline, 2weeks, 7weeks and 4months post-PCI. Myocardial blush grade was used to determine success of myocardial reperfusion. In multivariate models sex, age, hs-CRP levels at baseline, NT-proBNP levels at baseline, ischemia time, heart rate, TIMI flow, and CK, CKMB and troponin AUC were included. Follow-up was complete until 4months. RESULTS: Baseline hs-CRP levels were 2.1mg/l (IQR 0.5-4.2mg/l). hs-CRP levels were associated with impaired reperfusion (OR 1.239, 95% CI 1.006-1.527) and remained higher compared to patients with normal reperfusion up to 2months after PCI (hs-CRP 1.9mg/l (IQR 0.9-3.7mg/l) versus 1.5mg/l (IQR 0.7-2.7mg/l), p=0.041). In multivariate analysis baseline hs-CRP levels remained independently associated with impaired reperfusion. In patients with impaired reperfusion, hs-CRP and NT-proBNP levels remained higher during 4months of follow-up. No correlation was observed between hs-CRP at baseline and left ventricular function at 4months. The number of events was small and we observed no differences in mortality. CONCLUSION: Increased hs-CRP levels at presentation are associated with impaired microvascular reperfusion after PCI in STEMI patients and remain higher until 2months follow-up.

Deficiency of the TLR4 analogue RP105 aggravates vein graft disease by inducing a pro-inflammatory response.

Venous grafts are often used to bypass occlusive atherosclerotic lesions; however, poor patency leads to vein graft disease. Deficiency of TLR4, an inflammatory regulator, reduces vein graft disease. Here, we investigate the effects of the accessory molecule and TLR4 analogue RadioProtective 105 (RP105) on vein graft disease. RP105 deficiency resulted in a 90% increase in vein graft lesion area compared to controls. In a hypercholesterolemic setting (LDLr(-/-)/RP105(-/-) versus LDLr(-/-) mice), which is of importance as vein graft disease is usually characterized by excessive atherosclerosis, total lesion area was not affected. However we did observe an increased number of unstable lesions and intraplaque hemorrhage upon RP105 deficiency. In both setups, lesional macrophage content, and lesional CCL2 was increased. In vitro, RP105(-/-) smooth muscle cells and mast cells secreted higher levels of CCL2. In conclusion, aggravated vein graft disease caused by RP105 deficiency results from an increased local inflammatory response.

RP105 deficiency attenuates early atherosclerosis via decreased monocyte influx in a CCR2 dependent manner.

OBJECTIVE: Toll like receptor 4 (TLR4) plays a key role in inflammation and previously it was established that TLR4 deficiency attenuates atherosclerosis. RadioProtective 105 (RP105) is a structural homolog of TLR4 and an important regulator of TLR4 signaling, suggesting that RP105 may also be an important effector in atherosclerosis. We thus aimed to determine the role of RP105 in atherosclerotic lesion development using RP105 deficient mice on an atherosclerotic background. METHODS AND RESULTS: Atherosclerosis was induced in Western-type diet fed low density lipoprotein receptor deficient (LDLr(-/-)) and LDLr/RP105 double knockout (LDLr(-/-)/RP105(-/-)) mice by means of perivascular carotid artery collar placement. Lesion size was significantly reduced by 58% in LDLr(-/-)/RP105(-/-) mice, and moreover, plaque macrophage content was markedly reduced by 40%. In a model of acute peritonitis, monocyte influx was almost 3-fold reduced in LDLr(-/-)/RP105(-/-) mice (P = 0.001), while neutrophil influx remained unaltered, suggestive of an altered migratory capacity of monocytes upon deletion of RP105. Interestingly, in vitro stimulation of monocytes with LPS induced a downregulation of CCR2, a chemokine receptor crucially involved in monocyte influx to atherosclerotic lesions, which was more pronounced in LDLr(-/-)/RP105(-/-) monocytes as compared to LDLr(-/-) monocytes. CONCLUSION: We here show that RP105 deficiency results in reduced early atherosclerotic plaque development with a marked decrease in lesional macrophage content, which may be due to disturbed migration of RP105 deficient monocytes resulting from CCR2 downregulation.

TLR4 accessory molecule RP105 (CD180) regulates monocyte-driven arteriogenesis in a murine hind limb ischemia model.

AIMS: We investigated the role of the TLR4-accessory molecule RP105 (CD180) in post-ischemic neovascularization, i.e. arteriogenesis and angiogenesis. TLR4-mediated activation of pro-inflammatory Ly6Chi monocytes is crucial for effective neovascularization. Immunohistochemical analyses revealed that RP105+ monocytes are present in the perivascular space of remodeling collateral arterioles. As RP105 inhibits TLR4 signaling, we hypothesized that RP105 deficiency would lead to an unrestrained TLR4-mediated inflammatory response and hence to enhanced blood flow recovery after ischemia. METHODS AND RESULTS: RP105-/- and wild type (WT) mice were subjected to hind limb ischemia and blood flow recovery was followed by Laser Doppler Perfusion Imaging. Surprisingly, we found that blood flow recovery was severely impaired in RP105-/- mice. Immunohistochemistry showed that arteriogenesis was reduced in these mice compared to the WT. However, both in vivo and ex vivo analyses showed that circulatory pro-arteriogenic Ly6Chi monocytes were more readily activated in RP105-/- mice. FACS analyses showed that Ly6Chi monocytes became activated and migrated to the affected muscle tissues in WT mice following induction of hind limb ischemia. Although Ly6Chi monocytes were readily activated in RP105-/- mice, migration into the ischemic tissues was hampered and instead, Ly6Chi monocytes accumulated in their storage compartments, bone marrow and spleen, in RP105-/- mice. CONCLUSIONS: RP105 deficiency results in an unrestrained inflammatory response and monocyte over-activation, most likely due to the lack of TLR4 regulation. Inappropriate, premature systemic activation of pro-inflammatory Ly6Chi monocytes results in reduced infiltration of Ly6Chi monocytes in ischemic tissues and in impaired blood flow recovery.

TLR accessory molecule RP105 (CD180) is involved in post-interventional vascular remodeling and soluble RP105 modulates neointima formation.

BACKGROUND: RP105 (CD180) is TLR4 homologue lacking the intracellular TLR4 signaling domain and acts a TLR accessory molecule and physiological inhibitor of TLR4-signaling. The role of RP105 in vascular remodeling, in particular post-interventional remodeling is unknown. METHODS AND RESULTS: TLR4 and RP105 are expressed on vascular smooth muscle cells (VSMC) as well as in the media of murine femoral artery segments as detected by qPCR and immunohistochemistry. Furthermore, the response to the TLR4 ligand LPS was stronger in VSMC from RP105(-/-) mice resulting in a higher proliferation rate. In RP105(-/-) mice femoral artery cuff placement resulted in an increase in neointima formation as compared to WT mice (4982 ± 974 µm(2) vs.1947 ± 278 µm(2),p = 0.0014). Local LPS application augmented neointima formation in both groups, but in RP105(-/-) mice this effect was more pronounced (10316±1243 µm(2) vs.4208 ± 555 µm(2),p = 0.0002), suggesting a functional role for RP105. For additional functional studies, the extracellular domain of murine RP105 was expressed with or without its adaptor protein MD1 and purified. SEC-MALSanalysis showed a functional 2∶2 homodimer formation of the RP105-MD1 complex. This protein complex was able to block the TLR4 response in whole blood ex-vivo. In vivo gene transfer of plasmid vectors encoding the extracellular part of RP105 and its adaptor protein MD1 were performed to initiate a stable endogenous soluble protein production. Expression of soluble RP105-MD1 resulted in a significant reduction in neointima formation in hypercholesterolemic mice (2500 ± 573 vs.6581 ± 1894 µm(2),p<0.05), whereas expression of the single factors RP105 or MD1 had no effect. CONCLUSION: RP105 is a potent inhibitor of post-interventional neointima formation.

Blocking toll-like receptors 7 and 9 reduces postinterventional remodeling via reduced macrophage activation, foam cell formation, and migration.

OBJECTIVE: The role of toll-like receptors (TLRs) in vascular remodeling is well established. However, the involvement of the endosomal TLRs is unknown. Here, we study the effect of combined blocking of TLR7 and TLR9 on postinterventional remodeling and accelerated atherosclerosis. METHODS AND RESULTS: In hypercholesterolemic apolipoprotein E*3-Leiden mice, femoral artery cuff placement led to strong increase of TLR7 and TLR9 presence demonstrated by immunohistochemistry. Blocking TLR7/9 with a dual antagonist in vivo reduced neointimal thickening and foam cell accumulation 14 days after surgery by 65.6% (P=0.0079). Intima/media ratio was reduced by 64.5% and luminal stenosis by 62.8%. The TLR7/9 antagonist reduced the arterial wall inflammation, with reduced macrophage infiltration, decreased cytoplasmic high-mobility group box 1 expression, and altered serum interleukin-10 levels. Stimulation of cultured macrophages with TLR7 and TLR9 ligands enhanced tumor necrosis factor-α expression, which is decreased by TLR7/9 antagonist coadministration. Additionally, the antagonist abolished the TLR7/9-enhanced low-density lipoprotein uptake. The antagonist also reduced oxidized low-density lipoprotein-induced foam cell formation, most likely not via decreased influx but via increased efflux, because CD36 expression was unchanged whereas interleukin-10 levels were higher (36.1 ± 22.3 pg/mL versus 128.9 ± 6.6 pg/mL; P=0.008). CONCLUSIONS: Blocking TLR7 and TLR9 reduced postinterventional vascular remodeling and foam cell accumulation indicating TLR7 and TLR9 as novel therapeutic targets.

Future potential biomarkers for postinterventional restenosis and accelerated atherosclerosis.

New circulating and local arterial biomarkers may help the clinician with risk stratification or diagnostic assessment of patients and selecting the proper therapy for a patient. In addition, they may be used for follow-up and testing efficacy of therapy, which is not possible with current biomarkers. Processes leading to postinterventional restenosis and accelerated atherosclerosis are complex due to the many biological variables mediating the specific inflammatory and immunogenic responses involved. Adequate assessment of these processes requires different and more specific biomarkers. Postinterventional remodeling is associated with cell stress and tissue damage causing apoptosis, release of damage-associated molecular patterns and upregulation of specific cytokines/chemokines that could serve as suitable clinical biomarkers. Furthermore, plasma titers of pathophysiological process-related (auto)antibodies could aid in the identification of restenosis risk or lesion severity. This review provides an overview of a number of potential biomarkers selected on the basis of their role in the remodeling process.

Toll-like receptor 4 is involved in human and mouse vein graft remodeling, and local gene silencing reduces vein graft disease in hypercholesterolemic APOE*3Leiden mice.

OBJECTIVE: The goal of this study was to explore the role of Toll-like receptor 4 (TLR4) in vein graft remodeling and disease. METHODS AND RESULTS: First, expression of TLR4 was analyzed in freshly isolated human saphenous veins (huSV), in freshly isolated huSV ex vivo perfused in an extracorporeal circulation, or in huSV used as coronary vein grafts. Marked induction of focal TLR4 expression was observed in perfused fresh huSV. Moreover, TLR4 was abundantly present in lesions in fresh huSV or in intimal hyperplasia in coronary vein grafts. Second, mouse venous bypass grafting was performed. In grafts of hypercholesterolemic APOE*3Leiden mice, increased TLR4 mRNA and protein was detected over time by reverse transcription-polymerase chain reaction and immunohistochemistry. Furthermore, the local presence of the endogenous TLR4 ligands heat shock protein 60, high-mobility group box 1, tenascin-C, and biglycan in the grafts was demonstrated. TLR4 deficiency in C3H-Tlr4LPS-d (LPS indicates lipopolysaccharide) mice resulted in 48±12% less vein graft wall thickening (P=0.04) than in Balb/c controls. Moreover, local TLR4 gene silencing in hypercholesterolemic APOE*3Leiden mice using lentiviral short hairpin RNA against TLR4 administered perivascularly around vein grafts led to a 44±13% reduction of vessel wall thickening compared with controls (P=0.0059). CONCLUSIONS: These results indicate that TLR4 is involved in vein graft remodeling and can be used as a local therapeutic target against vein graft disease.

Lack of fibronectin-EDA promotes survival and prevents adverse remodeling and heart function deterioration after myocardial infarction.

RATIONALE: The extracellular matrix may induce detrimental inflammatory responses on degradation, causing adverse cardiac remodeling and heart failure. The extracellular matrix protein fibronectin-EDA (EIIIA; EDA) is upregulated after tissue injury and may act as a "danger signal" for leukocytes to cause adverse cardiac remodeling after infarction. OBJECTIVE: In the present study, we evaluated the role of EDA in regulation of postinfarct inflammation and repair after myocardial infarction. METHODS AND RESULTS: Wild-type and EDA(-/-) mice underwent permanent ligation of the left anterior coronary artery. Despite equal infarct size between groups (38.2±4.6% versus 38.2±2.9% of left ventricle; P=0.985), EDA(-/-) mice exhibited less left ventricular dilatation and enhanced systolic performance compared with wild-type mice as assessed by serial cardiac MRI measurements. In addition, EDA(-/-) mice exhibited reduced fibrosis of the remote area without affecting collagen production, cross-linking, and deposition in the infarct area. Subsequently, ventricular contractility and relaxation was preserved in EDA(-/-). At tissue level, EDA(-/-) mice showed reduced inflammation, metalloproteinase 2 and 9 activity, and myofibroblast transdifferentiation. Bone marrow transplantation experiments revealed that myocardium-induced EDA and not EDA from circulating cells regulates postinfarct remodeling. Finally, the absence of EDA reduced monocyte recruitment as well as monocytic Toll-like receptor 2 and CD49d expression after infarction. CONCLUSIONS: Our study demonstrated that parenchymal fn-EDA plays a critical role in adverse cardiac remodeling after infarction. Absence of fn-EDA enhances survival and cardiac performance by modulating matrix turnover and inflammation via leukocytes and fibroblasts after infarction.

In vivo suppression of vein graft disease by nonviral, electroporation-mediated, gene transfer of tissue inhibitor of metalloproteinase-1 linked to the amino terminal fragment of urokinase (TIMP-1.ATF), a cell-surface directed matrix metalloproteinase inhibitor.

BACKGROUND: Smooth muscle cell (SMC) migration and proliferation are important in the development of intimal hyperplasia, the major cause of vein graft failure. Proteases of the plasminogen activator (PA) system and of the matrix metalloproteinase (MMP) system are pivotal in extracellular matrix degradation and, by that, SMC migration. Previously, we demonstrated that inhibition of both protease systems simultaneously with viral gene delivery of the hybrid protein TIMP-1.ATF, consisting of the tissue inhibitor of metalloproteinase-1 (TIMP-1) and the receptor-binding amino terminal fragment (ATF) of urokinase, reduces SMC migration and neointima formation in an in vitro restenosis model using human saphenous vein cultures more efficiently than both protease systems separately. Because use of viral gene delivery is difficult in clinical application, this study used nonviral delivery of TIMP-1.ATF plasmid to reduce vein graft disease in a murine bypass model. Nonviral gene transfer by electroporation was used to avert major disadvantages of viral gene delivery, such as immune responses and short-term expression. METHODS: Plasmids encoding ATF, TIMP-1, TIMP-1.ATF, or luciferase, as a control, were injected and electroporated in both calf muscles of hypercholesterolemic apolipoprotein E3-Leiden (APOE*3Leiden) mice (n = 8). One day after electroporation, a venous interposition of a donor mouse was placed into the carotid artery of a recipient mouse. In this model, vein graft thickening develops with features of accelerated atherosclerosis. Vein grafts were harvested 4 weeks after electroporation and surgery, and histologic analysis of the vessel wall was performed. RESULTS: Electroporation-mediated overexpression of the plasmid vectors resulted in a prolonged expression of the transgenes and resulted in a significant reduction of vein graft thickening (ATF: 36% +/- 9%, TIMP-1: 49% +/- 5%, TIMP-1.ATF: 58% +/- 5%; P < .025). Although all constructs reduced vein graft thickening compared with the controls, the luminal area was best preserved in the TIMP-1.ATF-treated mice. CONCLUSION: Intramuscular electroporation of TIMP-1.ATF inhibits vein graft thickening in vein grafts in carotid arteries of hypercholesterolemic mice. Binding of TIMP-1.ATF hybrid protein to the u-PA receptor at the cell surface enhances the inhibitory effect of TIMP-1 on vein graft remodeling in vitro as well as in vivo and may be an effective strategy to prevent vein graft disease.