Mechanical strain triggers endothelial-to-mesenchymal transition of the endocardium in the immature heart.

BACKGROUND: Endothelial-to-mesenchymal-transition (EndMT) plays a major role in cardiac fibrosis, including endocardial fibroelastosis but the stimuli are still unknown. We developed an endothelial cell (EC) culture and a whole heart model to test whether mechanical strain triggers TGF-ß-mediated EndMT. METHODS: Isolated ECs were exposed to 10% uniaxial static stretch for 8 h (stretch) and TGF-ß-mediated EndMT was determined using the TGF-ß-inhibitor SB431542 (stretch + TGF-ß-inhibitor), BMP-7 (stretch + BMP-7) or losartan (stretch + losartan), and isolated mature and immature rats were exposed to stretch through a weight on the apex of the left ventricle. Immunohistochemical staining for double-staining with endothelial markers (VE-cadherin, PECAM1) and mesenchymal markers (αSMA) or transcription factors (SLUG/SNAIL) positive nuclei was indicative of EndMT. RESULTS: Stretch-induced EndMT in ECs expressed as double-stained ECs/total ECs (cells: 46 ± 13%; heart: 15.9 ± 2%) compared to controls (cells: 7 ± 2%; heart: 3.1 ± 0.1; p < 0.05), but only immature hearts showed endocardial EndMT. Inhibition of TGF-ß decreased the number of double-stained cells significantly, comparable to controls (cells/heart: control: 7 ± 2%/3.1 ± 0.1%, stretch: 46 ± 13%/15 ± 2%, stretch + BMP-7: 7 ± 2%/2.9 ± 0.1%, stretch + TGF-ß-inhibitor (heart only): 5.2 ± 1.3%, stretch + losartan (heart only): 0.89 ± 0.1%; p < 0.001 versus stretch). CONCLUSIONS: Endocardial EndMT is an age-dependent consequence of increased strain triggered by TGF- ß activation. Local inhibition through either rebalancing TGF-ß/BMP or with losartan was effective to block EndMT. IMPACT: Mechanical strain imposed on the immature LV induces endocardial fibroelastosis (EFE) formation through TGF-ß-mediated activation of endothelial-to-mesenchymal transition (EndMT) in endocardial endothelial cells but has no effect in mature hearts. Local inhibition through either rebalancing the TGF-ß/BMP pathway or with losartan blocks EndMT. Inhibition of endocardial EndMT with clinically applicable treatments may lead to a better outcome for congenital heart defects associated with EFE.

Endothelial-to-Mesenchymal Transition as Underlying Mechanism for the Formation of Double-Chambered Right Ventricle.

Double-chambered right ventricle (DCRV) is a progressive division of the right ventricular outflow tract (RVOT) often associated with a subaortic ventricular defect (VSD). The septation is caused by a mixture of hypertrophied muscle bundles and fibrous tissue, whereof the latter is of unclear pathogenesis. Our group has previously reported that flow disturbances lead to formation of fibroelastic tissue through a process called endothelial-to-mesenchymal transition (EndMT) but it is unclear whether the same mechanism exists in the RV. Tissue from patients undergoing repair of DCRV was examined to identify the histomorphological substrate of this tissue. Demographic and pre-/post-operative echocardiographic data were collected from nine patients undergoing surgery for DCRV. RVOTO tissue samples were histologically analyzed for myocardial hypertrophy, fibrosis, elastin content, and active EndMT (immunohistochemical double-staining for endothelial and mesenchymal markers and transcription factors Slug/Snail) and compared to four healthy controls. Indication for surgery were symptoms and progressive RVOT gradients. A highly turbulent flow jet through the RVOTO and VSD was observed in all patients with a preoperative median RVOT peak gradient of 77 mmHg (IQR 55.0-91.5), improved to 6 mmHg (IQR 4.5-17) postoperatively. Histological analysis revealed muscle and thick infiltratively growing fibroelastic tissue. EndMT was confirmed as underlying patho-mechanism of this fibroelastic tissue but the degree of myocardial hypertrophy was not different compared to controls (P = 0.08). This study shows for the first time that an invasive fibroelastic remodeling processes of the endocardium into the underlying myocardium through activation of EndMT contributes to the septation of the RVOT.

Human endothelial colony-forming cells provide trophic support for pluripotent stem cell-derived cardiomyocytes via distinctively high expression of neuregulin-1.

The search for a source of endothelial cells (ECs) with translational therapeutic potential remains crucial in regenerative medicine. Human blood-derived endothelial colony-forming cells (ECFCs) represent a promising source of autologous ECs due to their robust capacity to form vascular networks in vivo and their easy accessibility from peripheral blood. However, whether ECFCs have distinct characteristics with translational value compared to other ECs remains unclear. Here, we show that vascular networks generated with human ECFCs exhibited robust paracrine support for human pluripotent stem cell-derived cardiomyocytes (iCMs), significantly improving protection against drug-induced cardiac injury and enhancing engraftment at ectopic (subcutaneous) and orthotopic (cardiac) sites. In contrast, iCM support was notably absent in grafts with vessels lined by mature-ECs. This differential trophic ability was due to a unique high constitutive expression of the cardioprotective growth factor neuregulin-1 (NRG1). ECFCs, but not mature-ECs, were capable of actively releasing NRG1, which, in turn, reduced apoptosis and increased the proliferation of iCMs via the PI3K/Akt signaling pathway. Transcriptional silencing of NRG1 abrogated these cardioprotective effects. Our study suggests that ECFCs are uniquely suited to support human iCMs, making these progenitor cells ideal for cardiovascular regenerative medicine.

Endocardial fibroelastosis is caused by aberrant endothelial to mesenchymal transition.

RATIONALE: Endocardial fibroelastosis (EFE) is a unique form of fibrosis, which forms a de novo subendocardial tissue layer encapsulating the myocardium and stunting its growth, and which is typically associated with congenital heart diseases of heterogeneous origin, such as hypoplastic left heart syndrome. Relevance of EFE was only recently highlighted through the establishment of staged biventricular repair surgery in infant patients with hypoplastic left heart syndrome, where surgical removal of EFE tissue has resulted in improvement in the restrictive physiology leading to the growth of the left ventricle in parallel with somatic growth. However, pathomechanisms underlying EFE formation are still scarce, and specific therapeutic targets are not yet known. OBJECTIVE: Here, we aimed to investigate the cellular origins of EFE tissue and to gain insights into the underlying molecular mechanisms to ultimately develop novel therapeutic strategies. METHODS AND RESULTS: By utilizing a novel EFE model of heterotopic transplantation of hearts from newborn reporter mice and by analyzing human EFE tissue, we demonstrate for the first time that fibrogenic cells within EFE tissue originate from endocardial endothelial cells via aberrant endothelial to mesenchymal transition. We further demonstrate that such aberrant endothelial to mesenchymal transition involving endocardial endothelial cells is caused by dysregulated transforming growth factor beta/bone morphogenetic proteins signaling and that this imbalance is at least in part caused by aberrant promoter methylation and subsequent transcriptional suppression of bone morphogenetic proteins 5 and 7. Finally, we provide evidence that supplementation of exogenous recombinant bone morphogenetic proteins 7 effectively ameliorates endothelial to mesenchymal transition and experimental EFE in rats. CONCLUSIONS: In summary, our data point to aberrant endothelial to mesenchymal transition as a common denominator of infant EFE development in heterogeneous, congenital heart diseases, and to bone morphogenetic proteins 7 as an effective treatment for EFE and its restriction of heart growth.

Treating a 20 mm Hg gradient alleviates myocardial hypertrophy in experimental aortic coarctation.

BACKGROUND: Children with coarctation of the aorta (CoA) can have a hyperdynamic and remodeled left ventricle (LV) from increased afterload. Literature from an experimental model suggests the putative 20 mm Hg blood pressure gradient (BPG) treatment guideline frequently implemented in CoA studies may permit irreversible vascular changes. LV remodeling from pressure overload has been studied, but data are limited following correction and using a clinically representative BPG. MATERIALS AND METHODS: Rabbits underwent CoA at 10 weeks to induce a 20 mm Hg BPG using permanent or dissolvable suture thereby replicating untreated and corrected CoA, respectively. Cardiac function was evaluated at 32 weeks by magnetic resonance imaging using a spoiled cine GRE sequence (TR/TE/FA 8/2.9/20), 14 × 14-cm FOV, and 3-mm slice thickness. Images (20 frames/cycle) were acquired in 6-8 short axis views from the apex to the mitral valve annulus. LV volume, ejection fraction (EF), and mass were quantified. RESULTS: LV mass was elevated for CoA (5.2 ± 0.55 g) versus control (3.6 ± 0.16 g) and corrected (4.0 ± 0.44 g) rabbits, resulting in increased LV mass/volume ratio for CoA rabbits. A trend toward increased EF and stroke volume was observed but did not reach significance. Elevated EF by volumetric analysis in CoA rabbits was supported by concomitant increases in total aortic flow by phase-contrast magnetic resonance imaging. CONCLUSIONS: The indices quantified trended toward a persistent hyperdynamic LV despite correction, but differences were not statistically significant versus control rabbits. These findings suggest the current putative 20 mm Hg BPG for treatment may be reasonable from the LV's perspective.

Development of a Vascularized Heterotopic Neonatal Rat Heart Transplantation Model.

BACKGROUND/PURPOSE: Rodent adult-to-adult heterotopic heart transplantation is a well-established animal model, and the detailed surgical technique with several modifications has been previously described. In immature donor organ transplantation, however, the surgical technique needs to be revised given the smaller size and fragility of the donor graft. Here, we report our surgical technique for heterotopic abdominal (AHTx) and femoral (FHTx) neonatal rat heart transplantation based on an experience of over 300 cases. METHODS: Heterotopic heart transplantation was conducted in syngeneic Lewis rats. Neonatal rats (postnatal day 2-4) served as donors. AHTx was performed by utilizing the conventional adult-to-adult transplant method with specific modifications for optimal aortotomy and venous anastomosis. In the FHTx, the donor heart was vascularized by connecting the donor's aorta and pulmonary artery to the recipient's right femoral artery and vein, respectively, in an end-to-end manner. A specifically fashioned butterfly-shaped rubber sheet was used to align the target vessels properly. The transplanted graft was visually assessed for its viability and was accepted as a technical success when the viability met specific criteria. Successfully transplanted grafts were subject to further postoperative evaluation. Forty cases (AHTx and FHTx; n = 20 each) were compared regarding perioperative parameters and outcomes. RESULTS: Both models were technically feasible (success rate: AHTx 75% vs. FHTx 70%) by refining the conventional heterotopic transplant technique. Injury to the fragile donor aorta and congestion of the graft due to suboptimal venous connection were predominant causes of failure, leading to refractory bleeding and poor graft viability. Although the FHTx required significantly longer operation time and graft ischemic time, the in situ graft viabilities were comparable. The FHTx provided better postoperative monitoring as it enabled daily graft palpation and better echocardiographic visualization. CONCLUSIONS: We describe detailed surgical techniques for AHTx and FHTx while addressing neonatal donor-specific issues. Following our recommendations potentially reduces the learning curve to achieve reliable and reproducible results with these challenging animal models.

Pressure overload induces IL-18 and IL-18R expression, but markedly suppresses IL-18BP expression in a rabbit model. IL-18 potentiates TNF-α-induced cardiomyocyte death.

Recurrent or sustained inflammation plays a causal role in the development and progression of left ventricular hypertrophy (LVH) and its transition to failure. Interleukin (IL)-18 is a potent pro-hypertrophic inflammatory cytokine. We report that induction of pressure overload in the rabbit, by constriction of the descending thoracic aorta induces compensatory hypertrophy at 4weeks (mass/volume ratio: 1.7±0.11) and ventricular dilatation indicative of heart failure at 6weeks (mass/volume ratio: 0.7±0.04). In concordance with this, fractional shortening was preserved at 4weeks, but markedly attenuated at 6weeks. We cloned rabbit IL-18, IL-18Rα, IL-18Rß, and IL-18 binding protein (IL-18BP) cDNA, and show that pressure overload, while enhancing IL-18 and IL-18R expression in hypertrophied and failing hearts, markedly attenuated the level of expression of the endogenous IL-18 antagonist IL-18BP. Cyclical mechanical stretch (10% cyclic equibiaxial stretch, 1Hz) induced hypertrophy of primary rabbit cardiomyocytes in vitro and enhanced ANP, IL-18, and IL-18Rα expression. Further, treatment with rhIL-18 induced its own expression and that of IL-18Rα via AP-1 activation, and induced cardiomyocyte hypertrophy in part via PI3K/Akt/GATA4 signaling. In contrast, IL-18 potentiated TNF-α-induced cardiomyocyte death, and by itself induced cardiac endothelial cell death. These results demonstrate that pressure overload is associated with enhanced IL-18 and its receptor expression in hypertrophied and failingrabbit hearts. Since IL-18BP expression is markedly inhibited, our results indicate a positive amplification in IL-18 proinflammatory signaling during pressure overload, and suggest IL-18 as a potential therapeutic target in pathological hypertrophy and cardiac failure.

Intravenous Thrombolysis Before Thrombectomy Improves Functional Outcome After Stroke Independent of Reperfusion Grade.

BACKGROUND: We studied the association of bridging intravenous thrombolysis (IVT) before thrombectomy for anterior circulation large-vessel occlusion and functional outcome and scrutinized its dependence on grade of reperfusion and distal thrombus migration. METHODS AND RESULTS: We included consecutive patients with anterior circulation large-vessel occlusion from our prospective registry of thrombectomy-eligible patients treated from January 1, 2017 to January 1, 2023 at a tertiary stroke center in Germany in this retrospective cohort study. To evaluate the association of bridging IVT and functional outcome quantified via modified Rankin Scale score at 90 days we used multivariable logistic and lasso regression including interaction terms with grade of reperfusion quantified via modified Thrombolysis in Cerebral Infarction (mTICI) scale and distal thrombus migration adjusted for demographic and cardiovascular risk profiles, clinical and imaging stroke characteristics, onset-to-recanalization time and distal thrombus migration. We performed sensitivity analysis using propensity score matching. In our study population of 1000 thrombectomy-eligible patients (513 women; median age, 77 years [interquartile range, 67-84]), IVT emerged as a predictor of favorable functional outcome (modified Rankin Scale score, 0-2) independent of modified mTICI score (adjusted odds ratio, 0.49 [95% CI, 0.32-0.75]; P=0.001). In those who underwent thrombectomy (n=812), the association of IVT and favorable functional outcome was reproduced (adjusted odds ratio, 0.49 [95% CI, 0.31-0.74]; P=0.001) and was further confirmed on propensity score analysis, where IVT led to a 0.35-point decrease in 90-day modified Rankin Scale score (ß=-0.35 [95 CI%, -0.68 to 0.01]; P=0.04). The additive benefit of IVT remained independent of modified mTICI score (ß=-1.79 [95% CI, -3.43 to -0.15]; P=0.03) and distal thrombus migration (ß=-0.41 [95% CI, -0.69 to -0.13]; P=0.004) on interaction analysis. Consequently, IVT showed an additive association with functional outcome in the subpopulation of patients undergoing thrombectomy who achieved successful reperfusion (mTICI ≥2b; ß=-0.46 [95% CI, -0.74 to -0.17]; P=0.002) and remained beneficial in those with unsuccessful reperfusion (mTICI ≤2a; ß=-0.47 [95% CI, -0.96 to 0.01]; P=0.05). CONCLUSIONS: In thrombectomy-eligible patients with anterior circulation large-vessel occlusion, IVT improves functional outcome independent of grade of reperfusion and distal thrombus migration.

Pressure-overload hypertrophy of the developing heart reveals activation of divergent gene and protein pathways in the left and right ventricular myocardium.

Right ventricular (RV) and left ventricular (LV) myocardium differ in their pathophysiological response to pressure-overload hypertrophy. In this report we use microarray and proteomic analyses to identify pathways modulated by LV-aortic banding (AOB) and RV-pulmonary artery banding (PAB) in the immature heart. Newborn New Zealand White rabbits underwent banding of the descending thoracic aorta [LV-AOB; n = 6]. RV-PAB was achieved by banding the pulmonary artery (n = 6). Controls (n = 6 each) were sham-manipulated. After 4 (LV-AOB) and 6 (RV-PAB) wk recovery, the hearts were removed and matched RNA and proteins samples were isolated for microarray and proteomic analysis. Microarray and proteomic data demonstrate that in LV-AOB there is increased transcript expression levels for oxidative phosphorylation, mitochondria energy pathways, actin, ILK, hypoxia, calcium, and protein kinase-A signaling and increased protein expression levels of proteins for cellular macromolecular complex assembly and oxidative phosphorylation. In RV-PAB there is also an increased transcript expression levels for cardiac oxidative phosphorylation but increased protein expression levels for structural constituents of muscle, cardiac muscle tissue development, and calcium handling. These results identify divergent transcript and protein expression profiles in LV-AOB and RV-PAB and provide new insight into the biological basis of ventricular specific hypertrophy. The identification of these pathways should allow for the development of specific therapeutic interventions for targeted treatment and amelioration of LV-AOB and RV-PAB to ameliorate morbidity and mortality.

An animal model of endocardial fibroelastosis.

BACKGROUND: Hypoplastic left heart syndrome (HLHS) is one of the most common severe congenital cardiac anomalies, characterized by a marked hypoplasia of left-sided structures of the heart, which is commonly accompanied by a thick layer of fibroelastic tissue, termed endocardial fibroelastosis (EFE). Because human EFE develops only in fetal or neonatal hearts, and often in association with reduced blood flow, we sought to mimic these conditions by subjecting neonatal and 2-wk-old rat hearts to variations of the heterotopically transplanted heart model with either no intracavitary or normal flow and compare endocardium with human EFE tissue. MATERIALS AND METHODS: Hearts obtained from neonatal and 2-wk-old rats were heterotopically transplanted in young adult Lewis rats in a working (loaded) or nonworking (unloaded) mode. After 2-wk survival, hearts were explanted for histologic analysis by staining for collagen, elastin, and cellular elements. These sections were compared with human EFE tissue from HLHS. RESULTS: EFE, consisting of collagen and elastin with scarce cellular and vascular components, developed only in neonatal unloaded transplanted hearts and displayed the same histopathologic findings as EFE from patients with HLHS. Loaded hearts and 2-wk-old hearts did not show these alterations. CONCLUSIONS: This animal model for EFE will serve as a tool to study the mechanisms of EFE formation, such as fluid forces, in HLHS in a systematic manner. A better understanding of the underlying cause of the EFE formation in HLHS will help to develop novel treatment strategies to better preserve growth of the hypoplastic left ventricle.

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition.

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium. To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model. A neonatal rat heart is transplanted into an adolescent rat's abdomen and connected to the recipient's infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients. This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

No effect of short-term hypertension on bone matrix mineralization in a surgical animal model in immature rabbits.

Epidemiological studies show that arterial hypertension is associated with bone loss and an increased risk of fractures. Bone material properties are essential for bone strength. However, little is known about the effects of hypertension on bone matrix mineralization. Genetic animal models of hypertension are not ideal for studying bone matrix properties since these mutations may affect mineralization per se. The purpose of this study was to evaluate the effects of short-term hypertension on bone mineral density distribution (BMDD) using quantitative backscattered electron imaging in the proximal humerus in an established surgical model of pressure-overload cardiac hypertrophy in immature rabbits. Banding of the descending aorta was performed in 10-day-old rabbits (n = 4). Systolic blood pressure was elevated at all timepoints in the upper extremity but reached statistical significance at 5 and 6 weeks of age (+30.1% and +25.1 mm Hg; P < 0.05 each, respectively). Diastolic blood pressure was not affected. The left proximal humerus was harvested at 6 weeks of age, which is the maximum time in this animal model. Four non-operated, matched animals served as controls. Bone mineral density distribution parameters were determined in the epiphyseal and metaphyseal regions of the proximal humerus. The bone mineral density distribution parameters which describe the degree and heterogeneity of mineralization as well as the amount of low mineralized matrix showed no significant differences. Moreover no difference in bone length was found. Our study indicates that short-term elevation of blood pressure has no effects on bone matrix mineralization in this surgical model of pressure-overload cardiac hypertrophy in immature rabbits.

Case report: Active clinical manifestation of endocardial fibroelastosis in adolescence in a patient with mitral and aortic obstruction-histologic presence of endothelial-to-mesenchymal transformation.

This is the first description of active clinical manifestation of endocardial fibroelastosis (EFE) and remodeling of the endocardium via endothelial-to-mesenchymal transformation (EndMT) in an adolescent with Shone's variant hypoplastic left heart complex (HLHC) and a genetic heterozygous ABL1 variant. While EFE has not been typically associated HLHC or Shone's syndrome, in this patient flow alterations in the left ventricle (LV), combined with genetic alterations of intrinsic EndMT pathways led to active clinical manifestation of EFE in adolescence. This case emphasizes that new therapies for EFE might need to focus on molecular factors influenced by intrinsic and extrinsic stimuli of EndMT.

Autogenous mitochondria transplantation for treatment of right heart failure.

BACKGROUND: Right ventricular hypertrophy and failure are major causes of cardiac morbidity and mortality. A key event in the progression to right ventricular hypertrophy and failure is cardiomyocyte apoptosis due to mitochondrial dysfunction. We sought to determine whether localized intramyocardial injection of autologous mitochondria from healthy muscle treats heart failure. METHODS: Mitochondria transplanted from different sources were initially tested in cultured hypertrophic cardiomyocytes. A right ventricular hypertrophy/right ventricular failure model created through banding of the pulmonary artery in immature piglets was used for treatment with autologous mitochondria (pulmonary artery banded mitochondria injected/treated n = 6) from calf muscle, versus vehicle (pulmonary artery banded vehicle injected/treated n = 6) injected into the right ventricular free-wall, and compared with sham-operated controls (sham, n = 6). Animals were followed for 8 weeks by echocardiography (free-wall thickness, contractility), and dp/dt max was measured concomitantly with cardiomyocyte hypertrophy, fibrosis, and apoptosis at study end point. RESULTS: Internalization of mitochondria and adenosine triphosphate levels did not depend on the source of mitochondria. At 4 weeks, banded animals showed right ventricular hypertrophy (sham: 0.28 ± 0.01 cm vs pulmonary artery banding: 0.4 ± 0.02 cm wall thickness; P = .001), which further increased in pulmonary artery banded mitochondria injected/treated but declined in pulmonary artery banded vehicle injected/treated (0.47 ± 0.02 cm vs 0.348 ± 0.03 cm; P = .01). Baseline contractility was not different but was significantly reduced in pulmonary artery banded vehicle injected/treated compared with pulmonary artery banded mitochondria injected/treated and so was dp/dtmax. There was a significant difference in apoptotic cardiomyocyte loss and fibrosis in sham versus hypertrophied hearts with most apoptosis in pulmonary artery banded vehicle injected/treated hearts (sham: 1 ± 0.4 vs calf muscle vs vehicle: 13 ± 1.7; P = .001 and vs pulmonary artery banded mitochondria injected/treated: 8 ± 1.9, P = .01; pulmonary artery banded vehicle injected/treated vs pulmonary artery banded mitochondria injected/treated, P = .05). CONCLUSIONS: Mitochondrial transplantation allows for prolonged physiologic adaptation of the pressure-loaded right ventricular and preservation of contractility by reducing apoptotic cardiomyocyte loss.

Abnormal Flow Conditions Promote Endocardial Fibroelastosis Via Endothelial-to-Mesenchymal Transition, Which Is Responsive to Losartan Treatment.

Endocardial fibroelastosis (EFE) is defined by fibrotic tissue on the endocardium and forms partly through aberrant endothelial-to-mesenchymal transition. However, the pathologic triggers are still unknown. In this study, we showed that abnormal flow induces EFE partly through endothelial-to-mesenchymal transition in a rodent model, and that losartan can abrogate EFE development. Furthermore, we translated our findings to human endocardial endothelial cells, and showed that laminar flow promotes the suppression of genes associated with mesenchymal differentiation. These findings emphasize the role of flow in promoting EFE in endocardial endothelial cells and provide a novel potential therapy to treat this highly morbid condition.

Flow disturbances and the development of endocardial fibroelastosis.

OBJECTIVES: Endothelial-to-mesenchymal transition (EndMT) has been identified as the underlying mechanism of endocardial fibroelastosis (EFE) formation. The purpose of this study was to determine whether hemodynamic alterations due to valvar defects promote EndMT and whether age-specific structural changes affect ventricular diastolic compliance despite extensive surgical resection of EFE tissue. MATERIAL AND METHODS: We analyzed EFE tissue from 24 patients with hypoplastic left heart syndrome (HLHS) who underwent left ventricular (LV) rehabilitation surgery at Boston Children's Hospital between December 2011 and March 2018. Six patients with flow disturbances across the aortic valve and/or mitral valve but no HLHS diagnosis and macroscopic appearance of "EFE-like tissue" in the LV were included for comparison. All samples were examined for amount of collagen/elastin production and degradation, and presence of active EndMT by histologic analysis. RESULTS: EFE tissue from patients with and without HLHS consisted predominantly of elastin and collagen fibers. There was no alteration in degradation activity for collagen or elastin as shown by in situ zymography. Active EndMT was found in all patients with and without HLHS with flow disturbances ("EFE-like"). In patients with HLHS, EFE infiltrated into the underlying myocardium with increasing age. CONCLUSIONS: Patients with and without HLHS with flow disturbances due to stenotic or incompetent valves develop EndMT-derived fibrotic tissue covering the LV. When EFE recurs, it is directly associated with flow disturbances and switches to an infiltrative growth pattern with increasing age, leading to increased diastolic stiffness of the LV.

Flow disturbances and progression of endocardial fibroelastosis - a case report.

Endocardial fibroelastosis (EFE) is described as thickening of the endocardium and is associated with hypoplastic left heart syndrome (HLHS). The stimulus for EFE and the mechanism for recurrence and/or progression need to be investigated. In this report, we describe the case of a 4-year-old HLHS patient who underwent several surgeries with EFE resections due to recurrence of EFE. EFE recurrence was associated with flow disturbances due to valvar defects. At her latest follow-up 7 months after the last surgery, competent valves and no EFE were identified on all imaging study.

Alloreactivity and allorecognition of syngeneic and allogeneic mitochondria.

Previously, we have demonstrated that the transplantation of autologous mitochondria is cardioprotective. No immune or autoimmune response was detectable following the single injection of autologous mitochondria. To expand the therapeutic potential and safety of mitochondrial transplantation, we now investigate the immune response to single and serial injections of syngeneic and allogeneic mitochondria delivered by intraperitoneal injection. Our results demonstrate that there is no direct or indirect, acute or chronic alloreactivity, allorecognition or damage-associated molecular pattern molecules (DAMPs) reaction to single or serial injections of either syngeneic or allogeneic mitochondria.

Age-dependent expression of VEGF isoforms and receptors in the rabbit anterior cruciate ligament.

Vascular endothelial growth factor (VEGF) gene gives rise to several distinct isoforms of VEGF. Those isoforms differ in biochemical and biological properties, and it has been reported that their expression patterns are tissue and age specific as well. We investigated the expression levels of VEGF isoforms (VEGF121, VEGF165, VEGF183, VEGF189) and its receptors (VEGFR-1, flt-1 and VEGFR-2, flk-1/KDR) in the anterior cruciate ligament (ACL) of 2- to 3-week-, 2-month-, and 18-month-old New Zealand White rabbits using Sybr green Real-Time RT-PCR. VEGF isoforms and both receptors were expressed in the ACL at all investigated ages. VEGF121 was found to be the most abundant isoform at the ages under investigation, followed by VEGF165, VEGF189 and VEGF183. All isoforms showed decreased expression levels with age, however the larger membrane bound isoforms, VEGF183 and VEGF189, showed the most striking age-associated decrease in expression level. VEGFR-1 expression levels increased with age, while the expression level of VEGFR-2 expression was highest at 2-3 weeks and was significantly lower at 2 and 18 months of age. Distinct age-associated differences in the expression level of VEGF isoforms as well as their receptors suggest differential physiological functions during development, maturation and ageing of the ACL.

Vascular endothelial growth factor prevents apoptosis and preserves contractile function in hypertrophied infant heart.

BACKGROUND: Cardiac hypertrophy is an adaptive response to increased workload that, if unrelieved, leads to heart failure. It has been reported that cardiomyocyte apoptosis contributes to failure, and that vascular endothelial growth factor (VEGF) treatment of hypertrophied myocardium increases capillary density and improves myocardial perfusion. In this study we hypothesized that VEGF treatment reduces cardiomyocyte apoptosis and thereby preserves myocardial contractile function. METHODS AND RESULTS: Newborn rabbits underwent aortic banding. At 4 and 6 weeks of age, hypertrophied animals were treated with intrapericardial administration of recombinant VEGF protein. Three groups of animals were investigated: age-matched controls (C), untreated hypertrophied (H), and VEGF-treated hypertrophied hearts (T). Cardiomyocyte apoptosis was determined by TUNEL staining and PARP cleavage (immunoblotting of nuclear extracts) and cardiac function by transthoracic echocardiography. Death attributable to severe heart failure occurred in 14 of 43 untreated and 2 of 29 VEGF-treated animals (P<0.01). TUNEL-positive cardiomyocyte nuclei (n/1000 nuclei) were significantly increased in untreated hearts at 5 weeks (H: 10+/-1.8 versus T: 3+/-0.7) and at 7 weeks (H: 13+/-3.6 versus T: 5+/-1.5; P<0.05). Increased apoptosis in untreated hypertrophy was also confirmed by the presence of PARP cleavage (H: 74+/-7 versus T: 41+/-4 arbitrary densitometry units; P<0.05). VEGF treatment preserved left ventricular mass, prevented dilation (T: 1.01+/-0.06 versus H: 0.77+/-0.07; P<0.05), and preserved contractility indices compared with untreated hearts. CONCLUSIONS: Lack of adaptive capillary growth impairs myocardial perfusion and substrate delivery in hypertrophying myocardium. VEGF treatment reduces myocardial apoptosis and prolongs survival in a model of severe progressive left ventricular hypertrophy. Promoting capillary growth with VEGF reduces apoptosis, preserves myocardial contractile function, and delays the onset of failure in pressure-loaded infant myocardium.