Chronic endothelial dopamine receptor stimulation improves endothelial function and hemodynamics in autosomal dominant polycystic kidney disease.

Altered polycystin-mediated endothelial flow mechanosensitivity contributes to the development of hypertension and cardiovascular complications in patients with autosomal dominant polycystic kidney disease (ADPKD). Stimulation of endothelial type 5 dopamine receptors (DR5) can acutely compensate for the endothelial consequences of polycystin deficiency, but the chronic impact of this approach must be evaluated in ADPKD. Nineteen patients with ADPKD on standard of care therapy were randomized to receive a 2-month treatment with the DR agonist rotigotine using transdermal patches, nine at 2 mg/24hours and ten at 4 mg/24hours or while ten were untreated. Rotigotine at the dose of 4 mg/24hours significantly increased nitric oxide release (nitrite levels from 10±30 to 46±34 nmol/L) and radial artery endothelium-dependent flow-mediated dilatation (from 16.4±6.3 to 22.5±7.3%) in response to hand skin heating. Systemic hemodynamics were not significantly modified but aplanation tonometry showed that rotigotine at 4 mg/24hours reduced aortic augmentation index and pulse pressure without affecting carotid-to femoral pulse wave velocity. Plasma creatinine and urea, urinary cyclic AMP, which contributes to cyst growth in ADPKD and copeptin, a surrogate marker of vasopressin, were not affected by rotigotine. In mice with a specific deletion of polycystin-1 in endothelial cells, chronic infusion of the peripheral DR5 agonist fenoldopam also improved mesenteric artery flow-mediated dilatation and reduced blood pressure. Thus, our study demonstrates that in patients with ADPKD, chronic administration of rotigotine improves conduit artery endothelial function through the restoration of flow-induced nitric oxide release as well as hemodynamics suggesting that endothelial DR5 activation may represent a promising pharmacological approach to prevent cardiovascular complications of ADPKD.

A Supraceliac Aortic Cross Clamping Model to Explore Remote Lung Injury and the Endothelial Glycocalyx.

BACKGROUND: We hypothesized that supraceliac aortic cross clamping could induce lung injury mediated by an inflammatory ischemia-reperfusion (IR) trigger. We aimed to characterize glycocalyx (GCX), a component of endothelial membrane, participating to remote lung injury. METHODS: Rats underwent supraceliac aortic cross clamping for 40 min and were sacrificed at 0, 3, 6, and 24 hr of reperfusion (n = 10/group). Each group was compared to sham (n = 6/group). GCX products (syndecan-1 [Sdc-1] and heparan sulfate [HS]), tumor necrosis factor-alpha (TNF-α), and interleukin-1ß (IL-1ß) were measured in plasma (enzyme-linked immunosorbent assay[ELISA]). Lungs were harvested for measurements of TNF-α, IL-1ß (polymerase chain reaction) and Sdc-1 (western blotting [WB]). Histologic lung injury scoring and pulmonary gravimetry were analyzed in a blinded manner. RESULTS: Plasmatic Sdc-1, HS, TNF-α, and IL-1ß reached peak levels at 3 hr. Levels were significantly higher in clamping groups than sham at 6 hr for Sdc-1, at 0 and 3 hr for HS, at 3 and 6 hr for TNF-α, and at 3 hr for IL-1ß. Lung TNF-α and Interleukin-1ß reached peak levels at 6 hr. Levels were significantly higher than sham at 6 and 24 hr for TNF-α and at 6 hr for IL-1ß. Lung Sdc-1 was lowest at 3 hr. Sdc-1 was not significantly different compared to sham at the different reperfusion times. At 3 hr, it was 0.27 ± 0.03 vs. 0.33 ± 0.02 (sham) (P = 0.09). Histopathologic scores at 6 and 24 hr were higher in clamping groups than sham. At 6 and 24 hr, it was higher for hemorrhage, polynuclear neutrophil (PNN) infiltration and intravascular leukocytes. Pulmonary edema was higher by gravimetry at 0 and 6 hr. CONCLUSIONS: Supra celiac aortic clamping causes early lung injury in relation with a systemic inflammatory response associated with altered GCX structure.

Priming of Cardiopulmonary Bypass with Human Albumin Decreases Endothelial Dysfunction after Pulmonary Ischemia-Reperfusion in an Animal Model.

The routine use of mechanical circulatory support during lung transplantation (LTx) is still controversial. The use of prophylactic human albumin (HA) or hypertonic sodium lactate (HSL) prime in mechanical circulatory support during LTx could prevent ischemia−reperfusion (IR) injuries and pulmonary endothelial dysfunction and thus prevent the development of pulmonary graft dysfunction. The objective was to investigate the impact of cardiopulmonary bypass (CPB) priming with HA and HSL compared to a CPB prime with Gelofusine (GF) on pulmonary endothelial dysfunction in a lung IR rat model. Rats were assigned to four groups: IR-CPB-GF group, IR-CPB-HA group, IR-CPB-HSL group and a sham group. The study of pulmonary vascular reactivity by wire myograph was the primary outcome. Glycocalyx degradation (syndecan-1 and heparan) was also assessed by ELISA and electron microscopy, systemic and pulmonary inflammation by ELISA (IL-1ß, IL-10, and TNF-α) and immunohistochemistry. Clinical parameters were evaluated. We employed a CPB model with three different primings, permitting femoral−femoral assistance with left pulmonary hilum ischemia for IR. Pulmonary endothelium-dependent relaxation to acetylcholine was significantly decreased in the IR-CPB-GF group (11.9 ± 6.2%) compared to the IR-CPB-HA group (52.8 ± 5.2%, p < 0.0001), the IR-CPB-HSL group (57.7 ± 6.3%, p < 0.0001) and the sham group (80.8 ± 6.5%, p < 0.0001). We did not observe any difference between the groups concerning glycocalyx degradation, and systemic or tissular inflammation. The IR-CPB-HSL group needed more vascular filling and developed significantly more pulmonary edema than the IR-CPB-GF group and the IR-CPB-HA group. Using HA as a prime in CPB during Ltx could decrease pulmonary endothelial dysfunction's IR-mediated effects. No effects of HA were found on inflammation.

Lymphatic and Immune Cell Cross-Talk Regulates Cardiac Recovery After Experimental Myocardial Infarction.

OBJECTIVE: Lymphatics play an essential pathophysiological role in promoting fluid and immune cell tissue clearance. Conversely, immune cells may influence lymphatic function and remodeling. Recently, cardiac lymphangiogenesis has been proposed as a therapeutic target to prevent heart failure after myocardial infarction (MI). We investigated the effects of gene therapy to modulate cardiac lymphangiogenesis post-MI in rodents. Second, we determined the impact of cardiac-infiltrating T cells on lymphatic remodeling in the heart. Approach and Results: Comparing adenoviral versus adeno-associated viral gene delivery in mice, we found that only sustained VEGF (vascular endothelial growth factor)-CC156S therapy, achieved by adeno-associated viral vectors, increased cardiac lymphangiogenesis, and led to reduced cardiac inflammation and dysfunction by 3 weeks post-MI. Conversely, inhibition of VEGF-C/-D signaling, through adeno-associated viral delivery of soluble VEGFR3 (vascular endothelial growth factor receptor 3), limited infarct lymphangiogenesis. Unexpectedly, this treatment improved cardiac function post-MI in both mice and rats, linked to reduced infarct thinning due to acute suppression of T-cell infiltration. Finally, using pharmacological, genetic, and antibody-mediated prevention of cardiac T-cell recruitment in mice, we discovered that both CD4+ and CD8+ T cells potently suppress, in part through interferon-γ, cardiac lymphangiogenesis post-MI. CONCLUSIONS: We show that resolution of cardiac inflammation after MI may be accelerated by therapeutic lymphangiogenesis based on adeno-associated viral gene delivery of VEGF-CC156S. Conversely, our work uncovers a major negative role of cardiac-recruited T cells on lymphatic remodeling. Our results give new insight into the interconnection between immune cells and lymphatics in orchestration of cardiac repair after injury.

Protein tyrosine phosphatase 1B inactivation limits aging-associated heart failure in mice.

We have previously shown that protein tyrosine phosphatase 1B (PTP1B) inactivation in mice [PTP1B-deficient (PTP1B-/-) mice] improves left ventricular (LV) angiogenesis, perfusion, remodeling, and function and limits endothelial dysfunction after myocardial infarction. However, whether PTP1B inactivation slows aging-associated cardiovascular dysfunction remains unknown. Wild-type (WT) and PTP1B-/- mice were allowed to age until 18 mo. Compared with old WT mice, in which aging increased the LV mRNA expression of PTP1B, old PTP1B-/- mice had 1) reduced cardiac hypertrophy with decreased LV mRNA levels of hypertrophic markers and atrial and brain natriuretic peptides, 2) lower LV fibrosis (collagen: 16 ± 3% in WT mice and 5 ± 3% in PTP1B-/- mice, P < 0.001) with decreased mRNA levels of transforming growth-factor-ß1 and matrix metalloproteinase-2, and 3) higher LV capillary density and lower LV mRNA level of hypoxic inducible factor-1α, which was associated over time with a higher rate of proangiogenic M2 type macrophages and a stable LV mRNA level of VEGF receptor-2. Echocardiography revealed an age-dependent LV increase in end-diastolic volume in WT mice together with alterations of fractional shortening and diastole (transmitral Doppler E-to-A wave ratio). Invasive hemodynamics showed better LV systolic contractility and better diastolic compliance in old PTP1B-/- mice (LV end-systolic pressure-volume relation: 13.9 ± 0.9 in WT mice and 18.4 ± 1.6 in PTP1B-/- mice; LV end-diastolic pressure-volume relation: 5.1 ± 0.8 mmHg/relative volume unit in WT mice and 1.2 ± 0.3 mmHg/relative volume unit in PTP1B-/- mice, P < 0.05). In addition, old PTP1B-/- mice displayed a reduced amount of LV reactive oxygen species. Finally, in isolated resistance mesenteric arteries, PTP1B inactivation reduced aging-associated endothelial dysfunction (flow-mediated dilatation: -0.4 ± 2.1% in WT mice and 8.2 ± 2.8% in PTP1B-/- mice, P < 0.05). We conclude that PTP1B inactivation slows aging-associated LV remodeling and dysfunction and reduces endothelial dysfunction in mesenteric arteries. NEW & NOTEWORTHY The present study shows that protein tyrosine phosphatase 1B inactivation in aged mice improves left ventricular systolic and diastolic function associated with reduced adverse cardiac remodeling (hypertrophy, fibrosis, and capillary rarefaction) and limits vascular endothelial dysfunction. This suggests that protein tyrosine phosphatase 1B inhibition could be an interesting treatment approach in age-related cardiovascular dysfunction.

Short- and long-term administration of the non-steroidal mineralocorticoid receptor antagonist finerenone opposes metabolic syndrome-related cardio-renal dysfunction.

AIM: To determine whether non-steroidal mineralocorticoid receptor (MR) antagonists oppose metabolic syndrome-related end-organ, i.e. cardiac, damage. MATERIALS AND METHODS: In Zucker fa/fa rats, a rat model of metabolic syndrome, we assessed the effects of the non-steroidal MR antagonist finerenone (oral 2 mg/kg/day) on left ventricular (LV) function, haemodynamics and remodelling (using echocardiography, magnetic resonance imaging and biochemical methods). RESULTS: Long-term (90 days) finerenone modified neither systolic blood pressure nor heart rate, but reduced LV end-diastolic pressure and LV end-diastolic pressure-volume relationship, without modifying LV end-systolic pressure and LV end-systolic pressure-volume relationship. Simultaneously, long-term finerenone reduced both LV systolic and diastolic diameters, associated with reductions in LV weight and LV collagen density, while proteinuria and renal nGAL expression were reduced. Short-term (7 days) finerenone improved LV haemodynamics and reduced LV systolic diameter, without modifying LV diastolic diameter. Moreover, short-term finerenone increased myocardial tissue perfusion and reduced myocardial reactive oxygen species, while plasma nitrite levels, an indicator of nitric oxide (NO) bio-availability, were increased. CONCLUSIONS: In rats with metabolic syndrome, the non-steroidal MR antagonist finerenone opposed metabolic syndrome-related diastolic cardiac dysfunction and nephropathy. This involved acute effects, such as improved myocardial perfusion, reduced oxidative stress/increased NO bioavailability, as well as long-term effects, such as modifications in the myocardial structure.

A 30-Minute Supraceliac Aortic Clamping in the Rat Causes Death Due to an Inflammatory Response and Pulmonary Lesions.

BACKGROUND: The treatment of thoracoabdominal aortic aneurysms through an open approach has general and pulmonary consequences of multiple etiologies. Our assumption was that the supraceliac aortic clamping needed for this operation causes a systemic inflammatory response associated with a pulmonary attack. METHODS: We developed a model of 30-min supraceliac aortic clamping in Wistar rats weighing 300 g. After 90 min of reperfusion, the rats were sacrificed. The effects on the digestive tract wall were analyzed by measurement of the mucosal thickness/total thickness ratio. The effects on the mesenteric endothelial function were determined by an ex situ measurement of the arterial pressure/volume curves (third branch). The systemic consequences of the procedure were analyzed by dosing tumor necrosis factor alpha (TNFα), interleukin (IL)1ß, and IL10 in the blood. The pulmonary consequences were analyzed by the measurement of macrophages, polymorphonuclear neutrophils (PNs), T lymphocyte infiltration, pulmonary apoptosis (TUNEL) and active caspase 3. The experimental scheme included 20 rats with ischemia-reperfusion (IR) and 20 control rats. An analysis of survival was carried out on 20 other rats (10 IR and 10 controls). RESULTS: The results were expressed as average ± standard error of the mean. The statistical tests were Student's t-test and Mann-Whitney test. This visceral IR model decreased the ratio of the thickness of the intestinal mucosa compared with that of the control rats (0.77 ± 0.008 vs. 0.82 ± 0.009 [P < 0.001]). This local effect was not accompanied by any mesenteric endothelial dysfunction (P = 0.91). On a systemic level, IR increased TNFα (37.9 ± 1.5 vs. 28.2 ± 0.6 pg/mL; P < 0.0001), IL1ß (67.1 ± 9.8 vs. 22.5 ± 5.6 pg/mL; P < 0.001), and IL10 (753.3 ± 96 vs. 3.7 ± 1.7 pg/mL; P < 0.0001). As regards the lungs, IR increased the parenchymal cellular infiltration by macrophages (6.8 ± 0.8 vs. 4.5 ± 0.4 cells per field; P < 0.05) and PNs (7.4 ± 0.5 vs. 6.2 ± 03 cells per field; P < 0.05). There was no increase in the pulmonary cellular apoptosis measured by TUNEL (P = 0.77) or in the caspase 3 activity (P = 0.59). The mortality of the visceral IR rats was 100% at 36 hr vs. 0% in the animals without IR. CONCLUSIONS: This work showed that the inflammatory response to visceral IR had systemic and pulmonary effects which always results in the death in the rat.

Selective Stimulation of Cardiac Lymphangiogenesis Reduces Myocardial Edema and Fibrosis Leading to Improved Cardiac Function Following Myocardial Infarction.

BACKGROUND: The lymphatic system regulates interstitial tissue fluid balance, and lymphatic malfunction causes edema. The heart has an extensive lymphatic network displaying a dynamic range of lymph flow in physiology. Myocardial edema occurs in many cardiovascular diseases, eg, myocardial infarction (MI) and chronic heart failure, suggesting that cardiac lymphatic transport may be insufficient in pathology. Here, we investigate in rats the impact of MI and subsequent chronic heart failure on the cardiac lymphatic network. Further, we evaluate for the first time the functional effects of selective therapeutic stimulation of cardiac lymphangiogenesis post-MI. METHODS AND RESULTS: We investigated cardiac lymphatic structure and function in rats with MI induced by either temporary occlusion (n=160) or permanent ligation (n=100) of the left coronary artery. Although MI induced robust, intramyocardial capillary lymphangiogenesis, adverse remodeling of epicardial precollector and collector lymphatics occurred, leading to reduced cardiac lymphatic transport capacity. Consequently, myocardial edema persisted for several months post-MI, extending from the infarct to noninfarcted myocardium. Intramyocardial-targeted delivery of the vascular endothelial growth factor receptor 3-selective designer protein VEGF-CC152S, using albumin-alginate microparticles, accelerated cardiac lymphangiogenesis in a dose-dependent manner and limited precollector remodeling post-MI. As a result, myocardial fluid balance was improved, and cardiac inflammation, fibrosis, and dysfunction were attenuated. CONCLUSIONS: We show that, despite the endogenous cardiac lymphangiogenic response post-MI, the remodeling and dysfunction of collecting ducts contribute to the development of chronic myocardial edema and inflammation-aggravating cardiac fibrosis and dysfunction. Moreover, our data reveal that therapeutic lymphangiogenesis may be a promising new approach for the treatment of cardiovascular diseases.

Role of M2-like macrophage recruitment during angiogenic growth factor therapy.

Therapeutic angiogenesis has yet to fulfill its promise for the clinical treatment of ischemic diseases. Given the impact of macrophages during pathophysiological angiogenesis, we asked whether macrophages may similarly modulate vascular responses to targeted angiogenic therapies. Mouse matrigel plug assay and rat myocardial infarction (MI) model were used to assess angiogenic therapy with either VEGF-A or FGF-2 with HGF (F+H) delivered locally via albumin-alginate microcapsules. The infiltration of classical M1-type and alternative M2-like macrophages was assessed. Clodronate was used to prevent macrophage recruitment, and the VEGFR2 blocking antibody, DC101, to prevent VEGF-A signaling. At 3 weeks after matrigel implantation, the combination therapy (F+H) led to increased total, and specifically M2-like, macrophage infiltration versus control and VEGF-A plugs, correlating with the angiogenic response. In contrast, VEGF-A preferential recruited M1-type macrophages. In agreement with a direct role of M2-like macrophages in F+H-induced vessel growth, clodronate radically decreased angiogenesis. Further, DC101 reduced F+H-induced angiogenesis, without altering macrophage infiltration, revealing macrophage-derived VEGF-A as a crucial determinant of tissue responsiveness. Similarly, increased cardiac M2-like macrophage infiltration was found following F+H therapy post-MI, with strong correlation between macrophage levels and angiogenic and arteriogenic responses. In conclusion, M2-like macrophages play a decisive role, linked to VEGF-A production, in regulation of tissue responsiveness to angiogenic therapies including the combination of F+H. Our data suggest that future attempts at therapeutic revascularization in ischemic patients might benefit from coupling targeted growth factor delivery with either direct or indirect approaches to recruit pro-angiogenic macrophages in order to maximize therapeutic angiogenic/arteriogenic responses.

Impact of soluble epoxide hydrolase inhibition on early kidney damage in hyperglycemic overweight mice.

This study addressed the hypothesis that inhibition of the EETs degrading enzyme soluble epoxide hydrolase affords renal protection in the early stage of diabetic nephropathy. The renal effects of the sEH inhibitor t-AUCB (10mg/l in drinking water) were compared to those of the sulfonylurea glibenclamide (80mg/l), both administered for 8 weeks in FVB mice subjected to a high-fat diet (HFD, 60% fat) for 16 weeks. Mice on control chow diet (10% fat) and non-treated HFD mice served as controls. Compared with non-treated HFD mice, HFD mice treated with t-AUCB had a decreased EET degradation, as shown by their higher plasma EETs-to-DHETs ratio, and an increased EET production, as shown by the increase in EETs+DHETs levels, which was associated with induction of CYP450 epoxygenase expression. Both agents similarly reduced fasting glycemia but only t-AUCB prevented the increase in the urinary albumine-to-creatinine ratio in HFD mice. Histopathological analysis showed that t-AUCB reduced renal inflammation, which was associated with an increased mRNA expression of the NFκB inhibitor Iκ≡ and related decrease in MCP-1, COX2 and VCAM-1 expressions. Finally, there was a marginally significant increase in reactive oxygen species production in HFD mice, together with an enhanced NOX2 expression. Both agents did not modify these parameters but t-AUCB increased the expression of the antioxidant enzyme superoxide dismutase 1. These results demonstrate that, independently from its glucose-lowering effect, sEH inhibition prevents microalbuminuria and renal inflammation in overweight hyperglycemic mice, suggesting that this pharmacological strategy could be useful in the management of diabetic nephropathy.

Subtotal Nephrectomy Associated with a High-Phosphate Diet in Rats Mimics the Development of Calcified Aortic Valve Disease Associated with Chronic Renal Failure.

Introduction. This study addressed the hypothesis that subtotal nephrectomy associated with a high-phosphorus diet (5/6Nx + P) in rats represents a suitable animal model to mimic the cardiovascular consequences of chronic kidney disease (CKD) including calcified aortic valve disease (CAVD). Indeed, the latter contributes to the high morbidity and mortality of CKD patients and sorely lacks preclinical models for pathophysiological and pharmacological studies. Methods. Renal and cardiovascular function and structure were compared between sham-operated and 5/6 Nx rats + P 10 to 12 weeks after surgery. Results. As expected, 11 weeks after surgery, 5/6Nx + P rats developed CKD as demonstrated by their increase in plasma creatinine and urea nitrogen and decrease in glomerular filtration rate, estimated by using fluorescein-isothiocyanate-labelled sinistrin, anemia, polyuria, and polydipsia compared to sham-operated animals on a normal-phosphorus diet. At the vascular level, 5/6Nx + P rats had an increase in the calcium content of the aorta; a decrease in mesenteric artery dilatation in response to a stepwise increase in flow, illustrating the vascular dysfunction; and an increase in blood pressure. Moreover, immunohistology showed a marked deposition of hydroxyapatite crystals in the aortic valve of 5/6Nx + P rats. Echocardiography demonstrated that this was associated with a decrease in aortic valve cusp separation and an increase in aortic valve mean pressure gradient and in peak aortic valve velocity. Left-ventricular diastolic and systolic dysfunction as well as fibrosis were also present in 5/6Nx + P rats. Conclusion. This study demonstrates that 5/6Nx + P recapitulates the cardiovascular consequences observed in humans with CKD. In particular, the initiation of CAVD was shown, highlighting the interest of this animal model to study the mechanisms involved in the development of aortic stenosis and test new therapeutic strategies at an early stage of the disease.

Ezh2 emerges as an epigenetic checkpoint regulator during monocyte differentiation limiting cardiac dysfunction post-MI.

Epigenetic regulation of histone H3K27 methylation has recently emerged as a key step during alternative immunoregulatory M2-like macrophage polarization; known to impact cardiac repair after Myocardial Infarction (MI). We hypothesized that EZH2, responsible for H3K27 methylation, could act as an epigenetic checkpoint regulator during this process. We demonstrate for the first time an ectopic EZH2, and putative, cytoplasmic inactive localization of the epigenetic enzyme, during monocyte differentiation into M2 macrophages in vitro as well as in immunomodulatory cardiac macrophages in vivo in the post-MI acute inflammatory phase. Moreover, we show that pharmacological EZH2 inhibition, with GSK-343, resolves H3K27 methylation of bivalent gene promoters, thus enhancing their expression to promote human monocyte repair functions. In line with this protective effect, GSK-343 treatment accelerated cardiac inflammatory resolution preventing infarct expansion and subsequent cardiac dysfunction in female mice post-MI in vivo. In conclusion, our study reveals that pharmacological epigenetic modulation of cardiac-infiltrating immune cells may hold promise to limit adverse cardiac remodeling after MI.

Regulation and impact of cardiac lymphangiogenesis in pressure-overload-induced heart failure.

AIMS: Lymphatics are essential for cardiac health, and insufficient lymphatic expansion (lymphangiogenesis) contributes to development of heart failure (HF) after myocardial infarction. However, the regulation and impact of lymphangiogenesis in non-ischaemic cardiomyopathy following pressure-overload remains to be determined. Here, we investigated cardiac lymphangiogenesis following transversal aortic constriction (TAC) in C57Bl/6 and Balb/c mice, and in end-stage HF patients. METHODS AND RESULTS: Cardiac function was evaluated by echocardiography, and cardiac hypertrophy, lymphatics, inflammation, oedema, and fibrosis by immunohistochemistry, flow cytometry, microgravimetry, and gene expression analysis. Treatment with neutralizing anti-VEGFR3 antibodies was applied to inhibit cardiac lymphangiogenesis in mice. We found that VEGFR3-signalling was essential to prevent cardiac lymphatic rarefaction after TAC in C57Bl/6 mice. While anti-VEGFR3-induced lymphatic rarefaction did not significantly aggravate myocardial oedema post-TAC, cardiac immune cell levels were increased, notably myeloid cells at 3 weeks and T lymphocytes at 8 weeks. Moreover, whereas inhibition of lymphangiogenesis did not aggravate interstitial fibrosis, it increased perivascular fibrosis and accelerated development of left ventricular (LV) dilation and dysfunction. In clinical HF samples, cardiac lymphatic density tended to increase, although lymphatic sizes decreased, notably in patients with dilated cardiomyopathy. Similarly, comparing C57Bl/6 and Balb/c mice, lymphatic remodelling post-TAC was linked to LV dilation rather than to hypertrophy. The striking lymphangiogenesis in Balb/c was associated with reduced cardiac levels of macrophages, B cells, and perivascular fibrosis at 8 weeks post-TAC, as compared with C57Bl/6 mice that displayed weak lymphangiogenesis. Surprisingly, however, it did not suffice to resolve myocardial oedema, nor prevent HF development. CONCLUSIONS: We demonstrate for the first time that endogenous lymphangiogenesis limits TAC-induced cardiac inflammation and perivascular fibrosis, delaying HF development in C57Bl/6 but not in Balb/c mice. While the functional impact of lymphatic remodelling remains to be determined in HF patients, our findings suggest that under settings of pressure-overload poor cardiac lymphangiogenesis may accelerate HF development.

Endothelium-Specific Deficiency of Polycystin-1 Promotes Hypertension and Cardiovascular Disorders.

BACKGROUND: Autosomal dominant polycystic kidney disease is the most frequent hereditary kidney disease and is generally due to mutations in PKD1 and PKD2, encoding polycystins 1 and 2. In autosomal dominant polycystic kidney disease, hypertension and cardiovascular disorders are highly prevalent, but their mechanisms are partially understood. METHODS: Since endothelial cells express the polycystin complex, where it plays a central role in the mechanotransduction of blood flow, we generated a murine model with inducible deletion of Pkd1 in endothelial cells (Cdh5-CreERT2;Pkd1fl/fl) to specifically determine the role of endothelial polycystin-1 in autosomal dominant polycystic kidney disease. RESULTS: Endothelial deletion of Pkd1 induced endothelial dysfunction, as demonstrated by impaired flow-mediated dilatation of resistance arteries and impaired relaxation to acetylcholine, increased blood pressure and prevented the normal development of arteriovenous fistula. In experimental chronic kidney disease induced by subtotal nephrectomy, endothelial deletion of Pkd1 further aggravated endothelial dysfunction, vascular remodeling, and heart hypertrophy. CONCLUSIONS: Altogether, this study provides the first in vivo demonstration that specific deletion of Pkd1 in endothelial cells promotes endothelial dysfunction and hypertension, impairs arteriovenous fistula development, and potentiates the cardiovascular alterations associated with chronic kidney disease.

Mineralocorticoid receptor blockade with finerenone improves heart function and exercise capacity in ovariectomized mice.

AIMS: In post-menopausal women, incidence of heart failure with preserved ejection fraction is higher than in men. Hormonal replacement therapies did not demonstrate benefits. We tested whether the non-steroidal mineralocorticoid receptor antagonist finerenone limits the progression of heart failure in ovariectomized (OVX) mice with metabolic disorders. METHODS AND RESULTS: Ovariectomy was performed in 4-month-old mice, treated or not at 7 months old for 1 month with finerenone (Fine) 1 mg/kg/day. Left ventricular (LV) cardiac and coronary endothelial functions were assessed by echocardiography, catheterization, and myography. Blood pressure was measured by plethysmography. Insulin and glucose tolerance tests were performed. Exercise capacity and spontaneous activity were measured on treadmill and in combined indirect calorimetric cages equipped with voluntary running wheel. OVX mice presented LV diastolic dysfunction without modification of ejection fraction compared with controls (CTL), whereas finerenone improved LV filling pressure (LV end-diastolic pressure, mmHg: CTL 3.48 ± 0.41, OVX 6.17 ± 0.30**, OVX + Fine 3.65 ± 0.55† , **P < 0.01 vs. CTL, † P < 0.05 vs. OVX) and compliance (LV end-diastolic pressure-volume relation, mmHg/RVU: CTL 1.65 ± 0.42, OVX 4.77 ± 0.37***, OVX + Fine 2.87 ± 0.26†† , ***P < 0.001 vs. CTL, †† P < 0.01 vs. OVX). Acetylcholine-induced endothelial-dependent relaxation of coronary arteries was impaired in ovariectomized mice and improved by finerenone (relaxation, %: CTL 86 ± 8, OVX 38 ± 3**, OVX + Fine 83 ± 7†† , **P < 0.01 vs. CTL, †† P < 0.01 vs. OVX). Finerenone improved decreased ATP production by subsarcolemmal mitochondria after ovariectomy. Weight gain, increased blood pressure, and decreased insulin and glucose tolerance in OVX mice were improved by finerenone. The exercise capacity at race was diminished in untreated OVX mice only. Spontaneous activity measurements in ovariectomized mice showed decreased horizontal movements, reduced time spent in a running wheel, and reduced VO2 and VCO2 , all parameters improved by finerenone. CONCLUSIONS: Finerenone improved cardiovascular dysfunction and exercise capacity after ovariectomy-induced LV diastolic dysfunction with preserved ejection fraction.

Transient heart rate reduction improves acute decompensated heart failure-induced left ventricular and coronary dysfunction.

AIMS: Acute decompensated heart failure (ADHF), a live-threatening complication of heart failure (HF), associates a further decrease of the already by HF-impaired cardiac function with an increase in heart rate. We evaluated, using a new model of ADHF, whether heart rate reduction (HRR) opposes the acute decompensation-related aggravation of cardiovascular dysfunction. METHODS AND RESULTS: Cardiac output (echocardiography), cardiac tissue perfusion (magnetic resonance imaging), pulmonary wet weight, and in vitro coronary artery relaxation (Mulvany) were assessed 1 and 14 days after acute decompensation induced by salt-loading (1.8 g/kg, PO) in rats with well-established HF due to coronary ligation. HRR was induced by administration of the If current inhibitor S38844, 12 mg/kg PO twice daily for 2.5 days initiated 12 h or 6 days after salt-loading (early or delayed treatment, respectively). After 24 h, salt-loading resulted in acute decompensation, characterized by a reduction in cardiac output (HF: 130 ± 5 mL/min, ADHF: 105 ±  8 mL/min; P < 0.01), associated with a decreased myocardial perfusion (HF: 6.41 ± 0.53 mL/min/g, ADHF: 4.20 ± 0.11 mL/min/g; P < 0.01), a slight increase in pulmonary weight (HF: 1.68 ± 0.09 g, ADHF: 1.81 ± 0.15 g), and impaired coronary relaxation (HF: 55 ± 1% of pre-contraction at acetylcholine 4.5 10-5  M, ADHF: 27 ± 7 %; P < 0.01). Fourteen days after salt-loading, cardiac output only partially recovered (117 ± 5 mL/min; P < 0.05), while myocardial tissue perfusion (4.51 ± 0.44 mL/min; P < 0.01) and coronary relaxation (28 ± 4%; P < 0.01) remained impaired, but pulmonary weight further increased (2.06 ± 0.15 g, P < 0.05). Compared with untreated ADHF, HRR induced by S38844 improved cardiac output (125 ± 1 mL/min; P < 0.05), myocardial tissue perfusion (6.46 ± 0.42 mL/min/g; P < 0.01), and coronary relaxation (79 ± 2%; P < 0.01) as soon as 12 h after S38844 administration. These effects persisted beyond S38844 administration, illustrated by the improvements in cardiac output (130 ± 6 mL/min; P < 0.05), myocardial tissue perfusion (6.38 ± 0.48 mL/min/g; P < 0.01), and coronary relaxation (71 ± 4%; P < 0.01) at Day 14. S38844 did not modify pulmonary weight at Day 1 (1.78 ± 0.04 g) but tended to decrease pulmonary weight at Day 14 (1.80 ± 0.18 g). While delayed HRR induced by S38844 never improved cardiac function, early HRR rendered less prone to a second acute decompensation. CONCLUSIONS: In a model mimicking human ADHF, early, but not delayed, transient HRR induced by the If current inhibitor S38844 opposes acute decompensation by preventing the decompensated-related aggravation of cardiovascular dysfunction as well as the development of pulmonary congestion, and these protective effects persist beyond the transient treatment. Whether early transient HRR induced by If current inhibitors or other bradycardic agents, i.e. beta-blockers, exerts beneficial effects in human ADHF warrants further investigation.

Cardiopulmonary bypass increases endothelial dysfunction after pulmonary ischaemia-reperfusion in an animal model.

OBJECTIVES: Endothelial dysfunction during ischaemia-reperfusion (IR) is a major cause of primary graft dysfunction during lung transplantation. The routine use of cardiopulmonary bypass (CPB) during lung transplantation remains controversial. However, the contribution of CPB to pulmonary endothelial dysfunction remains unclear. The objective was to investigate the impact of CPB on endothelial dysfunction in a lung IR rat model. METHODS: Rats were allocated to 4 groups: (i) Sham, (ii) IR, (iii) CPB and (iv) IR-CPB. The primary outcome was the study of pulmonary vascular reactivity by wire myograph. We also assessed glycocalyx degradation by enzyme-linked immunosorbent assay and electron microscopy and both systemic and pulmonary inflammation by enzyme-linked immunosorbent assay and immunohistochemistry. Rats were exposed to 45 min of CPB and IR. We used a CPB model allowing femoro-femoral support with left pulmonary hilum ischaemia for IR. RESULTS: Pulmonary endothelium-dependent relaxation to acetylcholine was markedly reduced in the IR-CPB group (10.7 ± 9.1%) compared to the IR group (50.5 ± 5.2%, P < 0.001), the CPB group (54.1 ± 4.7%, P < 0.001) and the sham group (80.8 ± 6.7%, P < 0.001), suggesting that the association of pulmonary IR and CPB increases endothelial dysfunction. In IR-CPB, IR and CPB groups, vasorelaxation was completely abolished when inhibiting nitric oxide synthase, suggesting that this relaxation process was mainly mediated by nitric oxide. We observed higher syndecan-1 plasma levels in the IR-CPB group in comparison with the other groups, reflecting an increased degradation of glycocalyx. We also observed higher systemic inflammation in the IR-CPB group as shown by the increased plasma levels of IL-1ß, IL-10. CONCLUSIONS: CPB significantly increased the IR-mediated effects on pulmonary endothelial dysfunction. Therefore, the use of CPB during lung transplantation could be deleterious, by increasing endothelial dysfunction.

Impact of high-fat diet and vitamin D3 supplementation on aortic stenosis establishment in waved-2 epidermal growth factor receptor mutant mice.

OBJECTIVE: The use of animal models of aortic stenosis (AS) remains essential to further elucidate its pathophysiology and to evaluate new therapeutic strategies. The waved-2 mouse AS model has been proposed; data have indicated that while aortic regurgitation (AR) is effectively induced, development of AS is rare. We aimed to evaluate the effect of high-fat diet (HFD) and vitamin D3 supplementation in this model. METHODS: HFD and subcutaneous vitamin D3 injections were initiated at the age of 6 weeks until the age of 6 (n = 16, 6-month treatment group) and 9 (n = 11, 9-month treatment group) months. Twelve waved-2 mice without supplementation were used as control. Echocardiography was performed at 3, 6 and 9 months. Blood serum analysis (calcium, 1,25(OH)2D3 and cholesterol), histology and immunohistochemistry (CD-31, CD-68 and osteopontin) were evaluated at the end of the experiment (6 or 9 months). RESULTS: Total cholesterol and 1,25(OH)2D3 were significantly increased relative to the control group. HFD and vitamin D3 supplementation did result in improvements to the model, since AS was only detected in 6 (15.3%) mice (2 in the 3 groups) and AR was developed in the remaining animals. Echocardiographic parameters, fibrosis, thickness, inflammation and valvular calcification, were not significantly different between the 6-month treatment and control groups. Similar results were also observed in the 9-month treatment group. CONCLUSION: These results suggest that HFD and vitamin D3 supplementation have no effect in the waved-2 mouse model. This model essentially mimics AR and rarely AS. Further studies are needed to find a reliable animal model of AS.

Lebetin 2, a Snake Venom-Derived B-Type Natriuretic Peptide, Provides Immediate and Prolonged Protection against Myocardial Ischemia-Reperfusion Injury via Modulation of Post-Ischemic Inflammatory Response.

Myocardial infarction (MI) followed by left ventricular (LV) remodeling is the most frequent cause of heart failure. Lebetin 2 (L2), a snake venom-derived natriuretic peptide, exerts cardioprotection during acute myocardial ischemia-reperfusion (IR) ex vivo. However, its effects on delayed consequences of IR injury, including post-MI inflammation and fibrosis have not been defined. Here, we determined whether a single L2 injection exerts cardioprotection in IR murine models in vivo, and whether inflammatory response to ischemic injury plays a role in L2-induced effects. We quantified infarct size (IS), fibrosis, inflammation, and both endothelial cell and cardiomyocyte densities in injured myocardium and compared these values with those induced by B-type natriuretic peptide (BNP). Both L2 and BNP reduced IS, fibrosis, and inflammatory response after IR, as evidenced by decreased leukocyte and proinflammatory M1 macrophage infiltrations in the infarcted area compared to untreated animals. However, only L2 increased anti-inflammatory M2-like macrophages. L2 also induced a higher density of endothelial cells and cardiomyocytes. Our data show that L2 has strong, acute, prolonged cardioprotective effects in post-MI that are mediated, at least in part, by the modulation of the post-ischemic inflammatory response and especially, by the enhancement of M2-like macrophages, thus reducing IR-induced necrotic and fibrotic effects.

The IL-1ß Antibody Gevokizumab Limits Cardiac Remodeling and Coronary Dysfunction in Rats With Heart Failure.

This study reports preclinical data showing that the interleukin (IL)-1ß modulation is a new promising target in the pathophysiological context of heart failure. Indeed, in nondiabetic Wistar and diabetic Goto-Kakizaki rats with chronic heart failure induced by myocardial infarction, administration of the IL-1ß antibody gevokizumab improves 'surrogate' markers of survival (i.e., left ventricular remodeling, hemodynamics, and function as well as coronary function). However, whether IL-1ß modulation per se or in combination with standard treatments of heart failure improves long-term outcome in human heart failure remains to be determined.