Cripto favors chondrocyte hypertrophy via TGF-ß SMAD1/5 signaling during development of osteoarthritis.

Chondrocytes in mice developing osteoarthritis (OA) exhibit an aberrant response to the secreted cytokine transforming growth factor (TGF)-ß, consisting in a potentiation of intracellular signaling downstream of the transmembrane type I receptor kinase activin receptor-like kinase (ALK)1 against canonical TGF-ß receptor ALK5-mediated signaling. Unfortunately, the underlying mechanisms remain elusive. In order to identify novel druggable targets for OA, we aimed to investigate novel molecules regulating the ALK1/ALK5 balance in OA chondrocytes. We performed gene expression analysis of TGF-ß signaling modulators in joints from three different mouse models of OA and found an upregulated expression of the TGF-ß co-receptor Cripto (Tdgf1), which was validated in murine and human cartilage OA samples at the protein level. In vitro and ex vivo, elevated expression of Cripto favors the hypertrophic differentiation of chondrocytes, eventually contributing to tissue calcification. Furthermore, we found that Cripto participates in a TGF-ß-ALK1-Cripto receptor complex in the plasma membrane, thereby inducing catabolic SMAD1/5 signaling in chondrocytes. In conclusion, we demonstrate that Cripto is expressed in OA and plays a functional role promoting chondrocyte hypertrophy, thereby becoming a novel potential therapeutic target in OA, for which there is no efficient cure or validated biomarker. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

New calcification model for intact murine aortic valves.

Calcific aortic valve disease (CAVD) is a common progressive disease of the aortic valves, for which no medical treatment exists and surgery represents currently the only therapeutic solution. The development of novel pharmacological treatments for CAVD has been hampered by the lack of suitable test-systems, which require the preservation of the complex valve structure in a mechanically and biochemical controllable system. Therefore, we aimed at establishing a model which allows the study of calcification in intact mouse aortic valves by using the Miniature Tissue Culture System (MTCS), an ex vivo flow model for whole mouse hearts. Aortic valves of wild-type mice were cultured in the MTCS and exposed to osteogenic medium (OSM, containing ascorbic acid, ß-glycerophosphate and dexamethasone) or inorganic phosphates (PI). Osteogenic calcification occurred in the aortic valve leaflets that were cultured ex vivo in the presence of PI, but not of OSM. In vitro cultured mouse and human valvular interstitial cells calcified in both OSM and PI conditions, revealing in vitro-ex vivo differences. Furthermore, endochondral differentiation occurred in the aortic root of ex vivo cultured mouse hearts near the hinge of the aortic valve in both PI and OSM conditions. Dexamethasone was found to induce endochondral differentiation in the aortic root, but to inhibit calcification and the expression of osteogenic markers in the aortic leaflet, partly explaining the absence of calcification in the aortic valve cultured with OSM. The osteogenic calcifications in the aortic leaflet and the endochondral differentiation in the aortic root resemble calcifications found in human CAVD. In conclusion, we have established an ex vivo calcification model for intact wild-type murine aortic valves in which the initiation and progression of aortic valve calcification can be studied. The in vitro-ex vivo differences found in our studies underline the importance of ex vivo models to facilitate pre-clinical translational studies.

BMP Receptor Inhibition Enhances Tissue Repair in Endoglin Heterozygous Mice.

Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a severe vascular disorder caused by mutations in the TGFß/BMP co-receptor endoglin. Endoglin haploinsufficiency results in vascular malformations and impaired neoangiogenesis. Furthermore, HHT1 patients display an impaired immune response. To date it is not fully understood how endoglin haploinsufficient immune cells contribute to HHT1 pathology. Therefore, we investigated the immune response during tissue repair in Eng+/- mice, a model for HHT1. Eng+/- mice exhibited prolonged infiltration of macrophages after experimentally induced myocardial infarction. Moreover, there was an increased number of inflammatory M1-like macrophages (Ly6Chigh/CD206-) at the expense of reparative M2-like macrophages (Ly6Clow/CD206+). Interestingly, HHT1 patients also showed an increased number of inflammatory macrophages. In vitro analysis revealed that TGFß-induced differentiation of Eng+/- monocytes into M2-like macrophages was blunted. Inhibiting BMP signaling by treating monocytes with LDN-193189 normalized their differentiation. Finally, LDN treatment improved heart function after MI and enhanced vascularization in both wild type and Eng+/- mice. The beneficial effect of LDN was also observed in the hind limb ischemia model. While blood flow recovery was hampered in vehicle-treated animals, LDN treatment improved tissue perfusion recovery in Eng+/- mice. In conclusion, BMPR kinase inhibition restored HHT1 macrophage imbalance in vitro and improved tissue repair after ischemic injury in Eng+/- mice.

Prevention of progression of pulmonary hypertension by the Nur77 agonist 6-mercaptopurine: role of BMP signalling.

Pulmonary arterial hypertension (PAH) is a progressive fatal disease characterised by abnormal remodelling of pulmonary vessels, leading to increased vascular resistance and right ventricle failure. This abnormal vascular remodelling is associated with endothelial cell dysfunction, increased proliferation of smooth muscle cells, inflammation and impaired bone morphogenetic protein (BMP) signalling. Orphan nuclear receptor Nur77 is a key regulator of proliferation and inflammation in vascular cells, but its role in impaired BMP signalling and vascular remodelling in PAH is unknown.We hypothesised that activation of Nur77 by 6-mercaptopurine (6-MP) would improve PAH by inhibiting endothelial cell dysfunction and vascular remodelling.Nur77 expression is decreased in cultured pulmonary microvascular endothelial cells (MVECs) and lungs of PAH patients. Nur77 significantly increased BMP signalling and strongly decreased proliferation and inflammation in MVECs. In addition, conditioned medium from PAH MVECs overexpressing Nur77 inhibited the growth of healthy smooth muscle cells. Pharmacological activation of Nur77 by 6-MP markedly restored MVEC function by normalising proliferation, inflammation and BMP signalling. Finally, 6-MP prevented and reversed abnormal vascular remodelling and right ventricle hypertrophy in the Sugen/hypoxia rat model of severe angioproliferative PAH.Our data demonstrate that Nur77 is a critical modulator in PAH by inhibiting vascular remodelling and increasing BMP signalling, and activation of Nur77 could be a promising option for the treatment of PAH.

Endothelial Colony Forming Cells as an Autologous Model to Study Endothelial Dysfunction in Patients with a Bicuspid Aortic Valve.

Bicuspid aortic valve (BAV), the most common congenital heart defect, is associated with an increased prevalence of aortic dilation, aortic rupture and aortic valve calcification. Endothelial cells (ECs) play a major role in vessel wall integrity. Little is known regarding EC function in BAV patients due to lack of patient derived primary ECs. Endothelial colony forming cells (ECFCs) have been reported to be a valid surrogate model for several cardiovascular pathologies, thereby facilitating an in vitro system to assess patient-specific endothelial dysfunction. Therefore, the aim of this study was to investigate cellular functions in ECFCs isolated from BAV patients. Outgrowth and proliferation of ECFCs from patients with BAV (n = 34) and controls with a tricuspid aortic valve (TAV, n = 10) were determined and related to patient characteristics. Interestingly, we were only able to generate ECFCs from TAV and BAV patients without aortic dilation, and failed to isolate ECFC colonies from patients with a dilated aorta. Analyzing EC function showed that while proliferation, cell size and endothelial-to-mesenchymal transition were similar in TAV and BAV ECFCs, migration and the wound healing capacity of BAV ECFCs is significantly higher compared to TAV ECFCs. Furthermore, calcification is blunted in BAV compared to TAV ECFCs. Our results reveal ECs dysfunction in BAV patients and future research is required to unravel the underlying mechanisms and to further validate ECFCs as a patient-specific in vitro model for BAV.

Wild-type p53 inhibits pro-invasive properties of TGF-ß3 in breast cancer, in part through regulation of EPHB2, a new TGF-ß target gene.

The p53 tumor suppressor protein is primarily known for its important role in tumor suppression. In addition, p53 affects tumor cell migration, invasion, and epithelial-mesenchymal transition (EMT); processes also regulated by the transforming growth factor-ß (TGF-ß) signaling pathway. Here, we investigated the role of p53 in breast tumor cell invasion, migration, and EMT and examined the interplay of p53 with TGF-ß3 in these processes. MCF-10A1 and MCF-10CA1a breast cancer cells were treated with Nutlin-3 and TGF-ß3, and the effects on tumor cell migration and invasion were studied in transwell and 3D spheroid invasion assays. The effects of Nutlin-3 and TGF-ß3 on EMT were examined in NMuMG cells. To identify genes involved in TGF-ß-induced invasion that are modulated by p53, a Human Tumor Metastasis-specific RT-PCR array was performed. Verification of EPHB2 regulation by TGF-ß3 and p53 was performed on breast cancer tumor cell lines. We demonstrate that p53 inhibits basal and TGF-ß3-induced invasion, migration, and EMT in normal breast epithelial and breast cancer cells. Pharmacological activation of p53 inhibited induction of several TGF-ß3 targets involved in TGF-ß3-induced tumor cell invasion, i.e., matrix metallo proteinase (MMP)2, MMP9, and integrin ß 3 . The ephrin-type B receptor 2 (EPHB2) gene was identified as a new TGF-ß target important for TGF-ß3-mediated invasion and migration, whose transcriptional activation by TGF-ß3 is also inhibited by p53. The results show an intricate interplay between p53 and TGF-ß3 whereby p53 inhibits the TGF-ß3-induced expression of genes, e.g., EPHB2, to impede tumor cell invasion and migration.

HDMX-L is expressed from a functional p53-responsive promoter in the first intron of the HDMX gene and participates in an autoregulatory feedback loop to control p53 activity.

The p53 regulatory network is critically involved in preventing the initiation of cancer. In unstressed cells, p53 is maintained at low levels and is largely inactive, mainly through the action of its two essential negative regulators, HDM2 and HDMX. p53 abundance and activity are up-regulated in response to various stresses, including DNA damage and oncogene activation. Active p53 initiates transcriptional and transcription-independent programs that result in cell cycle arrest, cellular senescence, or apoptosis. p53 also activates transcription of HDM2, which initially leads to the degradation of HDMX, creating a positive feedback loop to obtain maximal activation of p53. Subsequently, when stress-induced post-translational modifications start to decline, HDM2 becomes effective in targeting p53 for degradation, thus attenuating the p53 response. To date, no clear function for HDMX in this critical attenuation phase has been demonstrated experimentally. Like HDM2, the HDMX gene contains a promoter (P2) in its first intron that is potentially inducible by p53. We show that p53 activation in response to a plethora of p53-activating agents induces the transcription of a novel HDMX mRNA transcript from the HDMX-P2 promoter. This mRNA is more efficiently translated than that expressed from the constitutive HDMX-P1 promoter, and it encodes a long form of HDMX protein, HDMX-L. Importantly, we demonstrate that HDMX-L cooperates with HDM2 to promote the ubiquitination of p53 and that p53-induced HDMX transcription from the P2 promoter can play a key role in the attenuation phase of the p53 response, to effectively diminish p53 abundance as cells recover from stress.

Oncogenic functions of hMDMX in in vitro transformation of primary human fibroblasts and embryonic retinoblasts.

BACKGROUND: In around 50% of all human cancers the tumor suppressor p53 is mutated. It is generally assumed that in the remaining tumors the wild-type p53 protein is functionally impaired. The two main inhibitors of p53, hMDM2 (MDM2) and hMDMX (MDMX/MDM4) are frequently overexpressed in wild-type p53 tumors. Whereas the main activity of hMDM2 is to degrade p53 protein, its close homolog hMDMX does not degrade p53, but it represses its transcriptional activity. Here we study the role of hMDMX in the neoplastic transformation of human fibroblasts and embryonic retinoblasts, since a high number of retinoblastomas contain elevated hMDMX levels. METHODS: We made use of an in vitro transformation model using a retroviral system of RNA interference and gene overexpression in primary human fibroblasts and embryonic retinoblasts. Consecutive knockdown of RB and p53, overexpression of SV40-small t, oncogenic HRasV12 and HA-hMDMX resulted in a number of stable cell lines representing different stages of the transformation process, enabling a comparison between loss of p53 and hMDMX overexpression. The cell lines were tested in various assays to assess their oncogenic potential. RESULTS: Both p53-knockdown and hMDMX overexpression accelerated proliferation and prevented growth suppression induced by introduction of oncogenic Ras, which was required for anchorage-independent growth and the ability to form tumors in vivo. Furthermore, we found that hMDMX overexpression represses basal p53 activity to some extent. Transformed fibroblasts with very high levels of hMDMX became largely resistant to the p53 reactivating drug Nutlin-3. The Nutlin-3 response of hMDMX transformed retinoblasts was intact and resembled that of retinoblastoma cell lines. CONCLUSIONS: Our studies show that hMDMX has the essential properties of an oncogene. Its constitutive expression contributes to the oncogenic phenotype of transformed human cells. Its main function appears to be p53 inactivation. Therefore, developing new drugs targeting hMDMX is a valid approach to obtain new treatments for a subset of human tumors expressing wild-type p53.

The Inflammatory Profile of CTEPH-Derived Endothelial Cells Is a Possible Driver of Disease Progression.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a form of pulmonary hypertension characterized by the presence of fibrotic intraluminal thrombi and causing obliteration of the pulmonary arteries. Although both endothelial cell (EC) dysfunction and inflammation are linked to CTEPH pathogenesis, regulation of the basal inflammatory response of ECs in CTEPH is not fully understood. Therefore, in the present study, we investigated the role of the nuclear factor (NF)-κB pro-inflammatory signaling pathway in ECs in CTEPH under basal conditions. Basal mRNA levels of interleukin (IL)-8, IL-1ß, monocyte chemoattractant protein-1 (MCP-1), C-C motif chemokine ligand 5 (CCL5), and vascular cell adhesion molecule-1 (VCAM-1) were upregulated in CTEPH-ECs compared to the control cells. To assess the involvement of NF-κB signaling in basal inflammatory activation, CTEPH-ECs were incubated with the NF-κB inhibitor Bay 11-7085. The increase in pro-inflammatory cytokines was abolished when cells were incubated with the NF-κB inhibitor. To determine if NF-κB was indeed activated, we stained pulmonary endarterectomy (PEA) specimens from CTEPH patients and ECs isolated from PEA specimens for phospho-NF-κB-P65 and found that especially the vessels within the thrombus and CTEPH-ECs are positive for phospho-NF-κB-P65. In summary, we show that CTEPH-ECs have a pro-inflammatory status under basal conditions, and blocking NF-κB signaling reduces the production of inflammatory factors in CTEPH-ECs. Therefore, our results show that the increased basal pro-inflammatory status of CTEPH-ECs is, at least partially, regulated through activation of NF-κB signaling and potentially contributes to the pathophysiology and progression of CTEPH.

Endothelium-derived stromal cells contribute to hematopoietic bone marrow niche formation.

Bone marrow stromal cells (BMSCs) play pivotal roles in tissue maintenance and regeneration. Their origins, however, remain incompletely understood. Here we identify rare LNGFR+ cells in human fetal and regenerative bone marrow that co-express endothelial and stromal markers. This endothelial subpopulation displays transcriptional reprogramming consistent with endothelial-to-mesenchymal transition (EndoMT) and can generate multipotent stromal cells that reconstitute the bone marrow (BM) niche upon transplantation. Single-cell transcriptomics and lineage tracing in mice confirm robust and sustained contributions of EndoMT to bone precursor and hematopoietic niche pools. Interleukin-33 (IL-33) is overexpressed in subsets of EndoMT cells and drives this conversion process through ST2 receptor signaling. These data reveal generation of tissue-forming BMSCs from mouse and human endothelial cells and may be instructive for approaches to human tissue regeneration.

Toward Biological Pacing by Cellular Delivery of Hcn2/SkM1.

Electronic pacemakers still face major shortcomings that are largely intrinsic to their hardware-based design. Radical improvements can potentially be generated by gene or cell therapy-based biological pacemakers. Our previous work identified adenoviral gene transfer of Hcn2 and SkM1, encoding a "funny current" and skeletal fast sodium current, respectively, as a potent combination to induce short-term biological pacing in dogs with atrioventricular block. To achieve long-term biological pacemaker activity, alternative delivery platforms need to be explored and optimized. The aim of the present study was therefore to investigate the functional delivery of Hcn2/SkM1 via human cardiomyocyte progenitor cells (CPCs). Nucleofection of Hcn2 and SkM1 in CPCs was optimized and gene transfer was determined for Hcn2 and SkM1 in vitro. The modified CPCs were analyzed using patch-clamp for validation and characterization of functional transgene expression. In addition, biophysical properties of Hcn2 and SkM1 were further investigated in lentivirally transduced CPCs by patch-clamp analysis. To compare both modification methods in vivo, CPCs were nucleofected or lentivirally transduced with GFP and injected in the left ventricle of male NOD-SCID mice. After 1 week, hearts were collected and analyzed for GFP expression and cell engraftment. Subsequent functional studies were carried out by computational modeling. Both nucleofection and lentiviral transduction of CPCs resulted in functional gene transfer of Hcn2 and SkM1 channels. However, lentiviral transduction was more efficient than nucleofection-mediated gene transfer and the virally transduced cells survived better in vivo. These data support future use of lentiviral transduction over nucleofection, concerning CPC-based cardiac gene delivery. Detailed patch-clamp studies revealed Hcn2 and Skm1 current kinetics within the range of previously reported values of other cell systems. Finally, computational modeling indicated that CPC-mediated delivery of Hcn2/SkM1 can generate stable pacemaker function in human ventricular myocytes. These modeling studies further illustrated that SkM1 plays an essential role in the final stage of diastolic depolarization, thereby enhancing biological pacemaker functioning delivered by Hcn2. Altogether these studies support further development of CPC-mediated delivery of Hcn2/SkM1 and functional testing in bradycardia models.

Supporting data on in vitro cardioprotective and proliferative paracrine effects by the human amniotic fluid stem cell secretome.

The data and information presented here refer to the research article entitled: "Reactivating endogenous mechanisms of cardiac regeneration via paracrine boosting with the human amniotic fluid stem cell secretome" (Balbi et al., 2019, Apr 04). This dataset illustrates the in vitro paracrine effect exerted by the human amniotic fluid stem cell secretome on rodent neonatal cardiomyocytes, human endothelial progenitors and different subsets of cardiac progenitor cells. Cytokine/chemokine profiling of the human amniotic fluid stem cell secretome is provided as well. This data can provide useful insights in regenerative medicine as demonstrating the in vitro cardioprotective and proliferative secretory paracrine potential of human fetal stem cells.

Reactivating endogenous mechanisms of cardiac regeneration via paracrine boosting using the human amniotic fluid stem cell secretome.

BACKGROUND: The adult mammalian heart retains residual regenerative capability via endogenous cardiac progenitor cell (CPC) activation and cardiomyocyte proliferation. We previously reported the paracrine cardioprotective capacity of human amniotic fluid-derived stem cells (hAFS) following ischemia or cardiotoxicity. Here we analyse the potential of hAFS secretome fractions for cardiac regeneration and future clinical translation. METHODS: hAFS were isolated from amniotic fluid leftover samples from prenatal screening. hAFS conditioned medium (hAFS-CM) was obtained following hypoxic preconditioning. Anti-apoptotic, angiogenic and proliferative effects were evaluated on rodent neonatal cardiomyocytes (r/mNVCM), human endothelial colony forming cells (hECFC) and human CPC. Mice undergoing myocardial infarction (MI) were treated with hAFS-CM, hAFS-extracellular vesicles (hAFS-EV), or EV-depleted hAFS-CM (hAFS-DM) by single intra-myocardial administration and evaluated in the short and long term. RESULTS: hAFS-CM improved mNVCM survival under oxidative and hypoxic damage, induced Ca2+-dependent angiogenesis in hECFC and triggered hCPC and rNVCM proliferation. hAFS-CM treatment after MI counteracted scarring, supported cardiac function, angiogenesis and cardiomyocyte cell cycle progression in the long term. hAFS-DM had no effect. hAFS-CM and hAFS-EV equally induced epicardium WT1+ CPC reactivation. Although no CPC cardiovascular differentiation was observed, our data suggests contribution to local angiogenesis by paracrine modulation. hAFS-EV alone were able to recapitulate all the beneficial effects exerted by hAFS-CM, except for stimulation of vessel formation. CONCLUSIONS: hAFS-CM and hAFS-EV can improve cardiac repair and trigger cardiac regeneration via paracrine modulation of endogenous mechanisms. While both formulations are effective in sustaining myocardial renewal, hAFS-CM retains higher pro-angiogenic potential, while hAFS-EV particularly enhances cardiac function.

Cardiac Progenitor Cell-Derived Extracellular Vesicles Reduce Infarct Size and Associate with Increased Cardiovascular Cell Proliferation.

Cell transplantation studies have shown that injection of progenitor cells can improve cardiac function after myocardial infarction (MI). Transplantation of human cardiac progenitor cells (hCPCs) results in an increased ejection fraction, but survival and integration are low. Therefore, paracrine factors including extracellular vesicles (EVs) are likely to contribute to the beneficial effects. We investigated the contribution of EVs by transplanting hCPCs with reduced EV secretion. Interestingly, these hCPCs were unable to reduce infarct size post-MI. Moreover, injection of hCPC-EVs did significantly reduce infarct size. Analysis of EV uptake showed cardiomyocytes and endothelial cells primarily positive and a higher Ki67 expression in these cell types. Yes-associated protein (YAP), a proliferation marker associated with Ki67, was also increased in the entire infarcted area. In summary, our data suggest that EV secretion is the driving force behind the short-term beneficial effect of hCPC transplantation on cardiac recovery after MI.

Development of Macrocycle Kinase Inhibitors for ALK2 Using Fibrodysplasia Ossificans Progressiva-Derived Endothelial Cells.

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare congenital form of heterotopic ossification (HO), caused by heterozygous mutations in the activin A type I receptor (ACVR1), that encodes the bone morphogenetic protein (BMP) type I receptor ALK2. These mutations enable ALK2 to induce downstream signaling in response to activins, thereby turning them into bone-inducing agents. To date, there is no cure for FOP. The further development of FOP patient-derived models may contribute to the discovery of novel biomarkers and therapeutic approaches. Nevertheless, this has traditionally been a challenge, as biopsy sampling often triggers HO. We have characterized peripheral blood-derived endothelial colony-forming cells (ECFCs) from three independent FOP donors as a new model for FOP. FOP ECFCs are prone to undergo endothelial-to-mesenchymal transition and exhibit increased ALK2 downstream signaling and subsequent osteogenic differentiation upon stimulation with activin A. Moreover, we have identified a new class of small molecule macrocycles with potential activity against ALK2 kinase. Finally, using FOP ECFCs, we have selected OD36 and OD52 as potent inhibitors with excellent kinase selectivity profiles that potently antagonize mutant ALK2 signaling and osteogenic differentiation. We expect that these results will contribute to the development of novel ALK2 clinical candidates for the treatment of FOP. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Nintedanib improves cardiac fibrosis but leaves pulmonary vascular remodelling unaltered in experimental pulmonary hypertension.

Aims: Pulmonary arterial hypertension (PAH) is associated with increased levels of circulating growth factors and corresponding receptors such as platelet derived growth factor, fibroblast growth factor and vascular endothelial growth factor. Nintedanib, a tyrosine kinase inhibitor targeting primarily these receptors, is approved for the treatment of patients with idiopathic pulmonary fibrosis. Our objective was to examine the effect of nintedanib on proliferation of human pulmonary microvascular endothelial cells (MVEC) and assess its effects in rats with advanced experimental pulmonary hypertension (PH). Methods and results: Proliferation was assessed in control and PAH MVEC exposed to nintedanib. PH was induced in rats by subcutaneous injection of Sugen (SU5416) and subsequent exposure to 10% hypoxia for 4 weeks (SuHx model). Four weeks after re-exposure to normoxia, nintedanib was administered once daily for 3 weeks. Effects of the treatment were assessed with echocardiography, right heart catheterization, and histological analysis of the heart and lungs. Changes in extracellular matrix production was assessed in human cardiac fibroblasts stimulated with nintedanib. Decreased proliferation with nintedanib was observed in control MVEC, but not in PAH patient derived MVEC. Nintedanib treatment did not affect right ventricular (RV) systolic pressure or total pulmonary resistance index in SuHx rats and had no effects on pulmonary vascular remodelling. However, despite unaltered pressure overload, the right ventricle showed less dilatation and decreased fibrosis, hypertrophy, and collagen type III with nintedanib treatment. This could be explained by less fibronectin production by cardiac fibroblasts exposed to nintedanib. Conclusion: Nintedanib inhibits proliferation of pulmonary MVECs from controls, but not from PAH patients. While in rats with experimental PH nintedanib has no effects on the pulmonary vascular pathology, it has favourable effects on RV remodelling.

Glycosylated Cell Surface Markers for the Isolation of Human Cardiac Progenitors.

The aim of stem cell therapy after cardiac injury is to replace damaged cardiac tissue. Human cardiac progenitor cells (CPCs) represent an interesting cell population for clinical strategies to treat cardiac disease and human CPC-specific antibodies would aid in the clinical implementation of cardiac progenitor-based cell therapy. However, the field of CPC biology suffers from the lack of human CPC-specific markers. Therefore, we raised a panel of monoclonal antibodies (mAb) against CPCs. Of this panel of antibodies, we show that mAb C1096 recognizes a progenitor-like population in the fetal and adult human heart and partially colocalize with reported CPC populations in vitro. Furthermore, mAb C1096 can be used to isolate a multipotent progenitor population from human heart tissue. Interestingly, the two lead candidates, mAb C1096 and mAb C19, recognize glycosylated residues on PECAM1 (platelet and endothelial cell adhesion molecule 1) and GRP78, respectively, and de-N-glycosylation significantly abolishes their binding. Thereby, this report describes new clinically applicable antibodies against human CPCs, and for the first time demonstrates the importance of glycosylated residues as CPCs specific markers.

Inhibiting DPP4 in a mouse model of HHT1 results in a shift towards regenerative macrophages and reduces fibrosis after myocardial infarction.

AIMS: Hereditary Hemorrhagic Telangiectasia type-1 (HHT1) is a genetic vascular disorder caused by haploinsufficiency of the TGFß co-receptor endoglin. Dysfunctional homing of HHT1 mononuclear cells (MNCs) towards the infarcted myocardium hampers cardiac recovery. HHT1-MNCs have elevated expression of dipeptidyl peptidase-4 (DPP4/CD26), which inhibits recruitment of CXCR4-expressing MNCs by inactivation of stromal cell-derived factor 1 (SDF1). We hypothesize that inhibiting DPP4 will restore homing of HHT1-MNCs to the infarcted heart and improve cardiac recovery. METHODS AND RESULTS: After inducing myocardial infarction (MI), wild type (WT) and endoglin heterozygous (Eng+/-) mice were treated for 5 days with the DPP4 inhibitor Diprotin A (DipA). DipA increased the number of CXCR4+ MNCs residing in the infarcted Eng+/- hearts (Eng+/- 73.17±12.67 vs. Eng+/- treated 157.00±11.61, P = 0.0003) and significantly reduced infarct size (Eng+/- 46.60±9.33% vs. Eng+/- treated 27.02±3.04%, P = 0.03). Echocardiography demonstrated that DipA treatment slightly deteriorated heart function in Eng+/- mice. An increased number of capillaries (Eng+/- 61.63±1.43 vs. Eng+/- treated 74.30±1.74, P = 0.001) were detected in the infarct border zone whereas the number of arteries was reduced (Eng+/- 11.88±0.63 vs. Eng+/- treated 6.38±0.97, P = 0.003). Interestingly, while less M2 regenerative macrophages were present in Eng+/- hearts prior to DipA treatment, (WT 29.88±1.52% vs. Eng+/- 12.34±1.64%, P<0.0001), DPP4 inhibition restored the number of M2 macrophages to wild type levels. CONCLUSIONS: In this study, we demonstrate that systemic DPP4 inhibition restores the impaired MNC homing in Eng+/- animals post-MI, and enhances cardiac repair, which might be explained by restoring the balance between the inflammatory and regenerative macrophages present in the heart.

Human fetal and adult epicardial-derived cells: a novel model to study their activation.

BACKGROUND: The epicardium, a cell layer covering the heart, plays an important role during cardiogenesis providing cardiovascular cell types and instructive signals, but becomes quiescent during adulthood. Upon cardiac injury the epicardium is activated, which includes induction of a developmental gene program, epithelial-to-mesenchymal transition (EMT) and migration. However, the response of the adult epicardium is suboptimal compared to the active contribution of the fetal epicardium to heart development. To understand the therapeutic value of epicardial-derived cells (EPDCs), a direct comparison of fetal and adult sources is paramount. Such analysis has been hampered by the lack of appropriate culture systems. METHODS: Human fetal and adult EPDCs were isolated from cardiac specimens obtained after informed consent. EPDCs were cultured in the presence of an inhibitor of the TGFß receptor ALK5. EMT was induced by stimulation with 1 ng/ml TGFß. PCR, immunofluorescent staining, scratch assay, tube formation assay and RT2-PCR for human EMT genes were performed to functionally characterize and compare fetal and adult EPDCs. RESULTS: In this study, a novel protocol is presented that allows efficient isolation of human EPDCs from fetal and adult heart tissue. In vitro, EPDCs maintain epithelial characteristics and undergo EMT upon TGFß stimulation. Although similar in several aspects, we observed important differences between fetal and adult EPDCs. Fetal and adult cells display equal migration abilities in their epithelial state. However, while TGFß stimulation enhanced adult EPDC migration, it resulted in a reduced migration in fetal EPDCs. Matrigel assays revealed the ability of adult EPDCs to form tube-like structures, which was absent in fetal cells. Furthermore, we observed that fetal cells progress through EMT faster and undergo spontaneous EMT when TGFß signaling is not suppressed, indicating that fetal EPDCs more rapidly respond to environmental changes. CONCLUSIONS: Our data suggest that fetal and adult EPDCs are in a different state of activation and that their phenotypic plasticity is determined by this activation state. This culture system allows us to establish the cues that determine epicardial activation, behavior, and plasticity and thereby optimize the adult response post-injury.

Exosomes from Cardiomyocyte Progenitor Cells and Mesenchymal Stem Cells Stimulate Angiogenesis Via EMMPRIN.

To date, cellular transplantation therapy has not yet fulfilled its high expectations for cardiac repair. A major limiting factor is lack of long-term engraftment of the transplanted cells. Interestingly, transplanted cells can positively affect their environment via secreted paracrine factors, among which are extracellular vesicles, including exosomes: small bi-lipid-layered vesicles containing proteins, mRNAs, and miRNAs. An exosome-based therapy will therefore relay a plethora of effects, without some of the limiting factors of cell therapy. Since cardiomyocyte progenitor cells (CMPC) and mesenchymal stem cells (MSC) induce vessel formation and are frequently investigated for cardiac-related therapies, the pro-angiogenic properties of CMPC and MSC-derived exosome-like vesicles are investigated. Both cell types secrete exosome-like vesicles, which are efficiently taken up by endothelial cells. Endothelial cell migration and vessel formation are stimulated by these exosomes in in vitro models, mediated via ERK/Akt-signaling. Additionally, these exosomes stimulated blood vessel formation into matrigel plugs. Analysis of pro-angiogenic factors revealed high levels of extracellular matrix metalloproteinase inducer (EMMPRIN). Knockdown of EMMPRIN on CMPCs leads to a diminished pro-angiogenic effect, both in vitro and in vivo. Therefore, CMPC and MSC exosomes have powerful pro-angiogenic effects, and this effect is largely mediated via the presence of EMMPRIN on exosomes.