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Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a severe vascular disorder caused by mutations in the TGFβ/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 TGFβ-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.

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.

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.

AimsHereditary Hemorrhagic Telangiectasia type-1 (HHT1) is a genetic vascular disorder caused by haploinsufficiency of the TGFβ 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 resultsAfter 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%, PConclusionsIn 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.

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 TGFβ receptor ALK5. EMT was induced by stimulation with 1 ng/ml TGFβ. 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 TGFβ 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 TGFβ 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 TGFβ 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.

Hereditary hemorrhagic telangiectasia (HHT) or Rendu–Osler–Weber disease is a rare genetic vascular disorder known for its endothelial dysplasia causing arteriovenous malformations and severe bleedings. HHT-1 and HHT-2 are the most prevalent variants and are caused by heterozygous mutations in endoglin and activin receptor-like kinase 1, respectively. An undervalued aspect of the disease is that HHT patients experience persistent inflammation. Although endothelial and mural cells have been the main research focus trying to unravel the mechanism behind the disease, wound healing is a process with a delicate balance between inflammatory and vascular cells. Inflammatory cells are part of the mononuclear cells (MNCs) fraction, and can, next to eliciting an immune response, also have angiogenic potential. This biphasic effect of MNC can hold a promising mechanism to further elucidate treatment strategies for HHT patients. Before MNC are able to contribute to repair, they need to home to and retain in ischemic and damaged tissue. Directed migration (homing) of MNCs following tissue damage is regulated by the stromal cell derived factor 1 (SDF1). MNCs that express the C-X-C chemokine receptor 4 (CXCR4) migrate toward the tightly regulated gradient of SDF1. This directed migration of monocytes and lymphocytes can be inhibited by dipeptidyl peptidase 4 (DPP4). Interestingly, MNC of HHT patients express elevated levels of DPP4 and show impaired homing toward damaged tissue. Impaired homing capacity of the MNCs might therefore contribute to the impaired angiogenesis and tissue repair observed in HHT patients. This review summarizes recent studies regarding the role of MNCs in the etiology of HHT and vascular repair, and evaluates the efficacy of DPP4 inhibition in tissue integrity and repair.

464 STAT4 deficiency exacerbates atherosclerosis by promoting mobilization of myeloid cells, polarization of M1 macrophages and formation of foam cells {#article-title-2} Background: Atherosclerosis (AS) is a chronic inflammatory disease of large and medium size vessels. Signal transducer and activator of transcription 4 (STAT4) has been reported to regulated the proliferation and differentiation of myeloid cells. However, the role of STAT4 in atherosclerotic progression is not well defined. Methods: We constructed APOE/STAT4 double knock out (DKO) mice via hybridization of ApoE-/- and STAT4-/- mice. 10 ApoE-/- mice (control) and 10 DKO mice were challenged with high-fat diet for 12 weeks. The extent of AS was determined by oil-red staining and HE staining. Plasma cholesterol, triglyceride and cytokines were assessed by ELISA. Changes in subsets of immune cells were evaluated by flow cytometry. Microarray analysis was applied to detect gene expressions while Western blot was used to assess protein levels. Results: Genetic deletion of STAT4 significantly exacerbated AS as evidenced by markedly increased oil-red-positive lipid-rich lesion in DKO mice accompanied by reduction of collagen fiber and increase of necrotic core lesion in plaques. Higher level of TC, TG and LDL-C in the serum and more abdominal fat were detected in DKO mice. Increased percentage of CD11b+Gr-1+ myeloid cells and Ly6Chigh M1 macrophage in peripheral blood, bone marrow and spleen of DKO mice suggested that STAT4 signal may play a critical role in regulating the proliferation and mobilization of myeloid cells and polarization of macrophages. To further explore the impact of STAT4 deficiency in myeloid cells, we isolated CD11b+ myeloid cells from bone marrows of ApoE-/- and DKO mice and incubated them with GM-CSF (60ng/ml) plus ox-LDL (60ug/ml). Enhanced differentiation of CD11b+Ly6Chigh macrophage and increase formation of foam cells were detected in DKO group. IFN-γ in the supernatant increased while IL-10 decreased in DKO group, indicating enhanced polarization of M1 macrophage from CD11b+ cells of DKO mice. Meanwhile, microarray data demonstrated that STAT4 KO increased expression levels of M1-related genes such as inducible nitric oxide synthase (iNos). Mechanistically, STAT4 deficiency significantly promoted the formation of foam cells by inhibiting of phosphatidylinositol-3 kinase/ serine-threonine kinases (PI3K/AKT) signal and consequently up-regulating of the expression of acyl coenzyme A: cholesterol acyltransferase-1 (ACAT-1), an enzyme that esterifies cholesterol and promotes its storage in macrophages. Conclusions: Our studies identified STAT4 as a regulator of the proliferation and differentiation of myeloid cells and atherogenesis. PI3K/AKT/ACAT-1 signaling was the molecular mechanisms of STAT4 functioning in the process. These findings points towards the development of STAT4 as a novel pharmacotherapeutic target for the treatment of atherosclerotic diseases and ApoE/STAT4 DKO mice with hyperlipidemia and hyper-inflammation as a novel mouse model more susceptible to atherosclerosis for future study. # 465 Effects of DPP4 inhibition on cardiac regeneration and macrophage balance in a mouse model of HHT-1 {#article-title-3} Background: Hereditary Hemorrhagic Telangiectasia type 1 (HHT-1) is a genetic dominant vascular disorder caused by haploinsufficiency of the TGFβ co-receptor Endoglin. Although the pathology of HHT-1 suggests that mainly vascular endothelial cells are involved, we have previously shown dysfunctional homing of mononuclear cells (MNC) towards the infarcted myocardium (MI). This is most likely due to enhanced expression of dipeptidyl peptidase-4 (DPP4). DPP4 inactivates SDF-1α, thereby inhibiting recruitment of CXCR4 expressing cells. Purpose: Our aim is to increase homing of the HHT-1 MNC and improve cardiac recovery and function in HHT-1 mice following MI, by inhibition of DPP4 activity. Methods: MI was induced in wildtype (WT) and endoglin heterozygous (eng+/-; as a model of HHT-1) mice by ligation of the left anterior descending (LAD) coronary artery, followed by 5 days of daily treatment with the DPP4 inhibitor Diprotin A (2.5mg/kg/day). The infarcted hearts were assessed at day 4 and at day 14 post MI. Results: DPP4 inhibition restored the number of MNC present in the infarcted hearts and significantly reduced infarct size (eng+/- 46.60±9.33% vs. eng+/- treated 27.02±3.04%, P=0.03), as measured by myocardial collagen formation. Analysis of cardiac function using ultrasound demonstrated that treatment of WT mice improved ejection fraction, however showed a slightly deteriorating effect in the eng+/- animals. Investigating the infarct borderzone, the number of capillaries increased (eng+/- 61.63±1.43 vs. eng+/- treated 74.30±1.74, P=0.001) while the number of arteries decreased (eng+/- 11.88±0.63 vs. eng+/- treated 6.38±0.97, P=0.003), suggesting that angiogenesis is upregulated, though the maturation of the new vessels is still impaired. Furthermore at day 4 post-MI, during the peak of inflammatory cell influx, Eng+/- mice show a significant decrease (WT 29.88±1.52 vs. eng+/- 12.34±1.64, P

Introduction: Endoglin is a co-receptor of Transforming Growth Factorβ (TGFβ) and is crucial for the formation of new blood vessels. Endoglin-haploinsuffiency is the leading cause of the severe vascular disease Hereditary hemorrhagic telangiectasia 1 (HHT-1). The aim of this study is to investigate whether endoglin haplo-insufficiency impairs macrophage polarization towards a regenerative phenotype to induce angiogenesis and tissue repair in ischemic disease. Materials and Methods: HHT-1 was studied in Eng+/- mice and in HHT-1 patients with endoglin mutation. In Eng+/- and Eng+/+ mice, angiogenesis and tissue repair were studied by the induction of hindlimb ischemia, myocardial infarction or in matrigel plugs. Macrophage phenotypes were screened after a differentiation assay in-vitro after stimulation with GM-CSF or in tissue sections with immuno-histochemistry. Results: Angiogenic capacity was impaired in Eng+/- mice, which was associated by the infiltration of macrophages. Ischemic muscle or heart sections of Eng+/- mice showed prolonged inflammation 7 days postischemia. We can explain this by an extended polarization of bone marrow-derived monocytes (BMM) from Eng+/- mice towards pro-inflammatory macrophage (CD11b+/LY6Chi/CD206-) at the expense of regenerative macrophages. In addition, stimulation of macrophages with TGFβ resulted in the polarization of the more regenerative phenotype only in WT BMM-cultures. This TGFβ response was blunted in macrophages from Eng+/- mice, but we were able to rescue it by the inhibition of the Bone Morphogenetic Protein (BMP)-pathway (by LDN-193189). Subsequently, LDN also improved blood flow recovery and myocardial function in Eng+/- mice after hind limb ischemia and MI, respectively. Interestingly, blood monocytes from HHT-1 patients show increased number of CD14+/CD16+ monocytes, which is associated with pro-inflammatory/non-regenerative phenotype. Discussion and Conclusions: Endoglin haplo-insufficiency results in defective tissue repair due to impaired polarization of regenerative macrophages, directed by TGFβ. The defective responses were rescued by the inhibition of another TGFβ familiy member, BMP, and thereby counterbalance its pathway. These results have major implications for the treatment of HHT-1 patients, as we show the same impaired macrophage polarization.