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-1β, 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

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>We made use of an <it>in vitro </it>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.</p> <p>Results</p> <p>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 <it>in vivo</it>. 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.</p> <p>Conclusions</p> <p>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.</p

HDMX-L is expressed from a functional P53-responsive promoter in the first intron of the HDMX gene, and participates in an auto-regulatory 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 upregulated 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

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

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

METACOHORTS for the study of vascular disease and its contribution to cognitive decline and neurodegeneration: an initiative of the Joint Programme for Neurodegenerative Disease Research

Dementia is a global problem and major target for health care providers. Although up to 45% of cases are primarily or partly due to cerebrovascular disease, little is known of these mechanisms or treatments because most dementia research still focuses on pure Alzheimer's disease. An improved understanding of the vascular contributions to neurodegeneration and dementia, particularly by small vessel disease, is hampered by imprecise data, including the incidence and prevalence of symptomatic and clinically “silent” cerebrovascular disease, long-term outcomes (cognitive, stroke, or functional), and risk factors. New large collaborative studies with long follow-up are expensive and time consuming, yet substantial data to advance the field are available. In an initiative funded by the Joint Programme for Neurodegenerative Disease Research, 55 international experts surveyed and assessed available data, starting with European cohorts, to promote data sharing to advance understanding of how vascular disease affects brain structure and function, optimize methods for cerebrovascular disease in neurodegeneration research, and focus future research on gaps in knowledge. Here, we summarize the results and recommendations from this initiative. We identified data from over 90 studies, including over 660,000 participants, many being additional to neurodegeneration data initiatives. The enthusiastic response means that cohorts from North America, Australasia, and the Asia Pacific Region are included, creating a truly global, collaborative, data sharing platform, linked to major national dementia initiatives. Furthermore, the revised World Health Organization International Classification of Diseases version 11 should facilitate recognition of vascular-related brain damage by creating one category for all cerebrovascular disease presentations and thus accelerate identification of targets for dementia prevention

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‐1β, 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

Glycosylated cell surface markers for the isolation of human cardiac progenitors

\u3cp\u3eThe 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.\u3c/p\u3

Additional file 1: Figure S1. of Human fetal and adult epicardial-derived cells: a novel model to study their activation

Isolation of human epicardium. (a) For isolation of EPDCs, the epicardium was removed from the underlying myocardium. (b) The isolated epicardial layer was positive for WT1 and negative for the myocardial markers cTnT and βMHC. In addition, the epicardial layer expressed, compared to myocardial tissue, low levels of the smooth muscle marker αSMA. This data was representative for three independent isolations, which all together showed the purity of the isolated epicardial layer. Figure S2. Human fetal and adult EPDCs express epicardium-related markers. (a) Summary of qRT-PCR analysis, indicating expression levels of genes reportedly expressed in the epicardium or EPDCs, showed that both fetal and adult EPDCs were positive for genes investigated. (b) WT1, (c) TBX18, and (d) TCF21 protein were present within the nucleus of both EPDC populations. Figure S3. Cultured human fetal and adult EPDCs do not display markers attributed to other heart-resident cell types. Gene expression was determined by qRT-PCR using several heart-related markers for (a-b) cardiac fibroblasts, (c) endocardium, (d) endothelial cells, (e) cardiac progenitor cells, (f) hematopoietic cells, and (g) mature cardiomyocytes. All data was shown as fold increase compared to their corresponding control cell type or tissue (N.D.: not detected). Figure S4. Fetal and adult EPDCs undergo EMT upon TGFβ stimulation. Validation of EMT was determined by qRT-PCR showing the downregulation of the epithelial genes ALDH1A2 and CDH1 in (a) fetal and (b) adult EPDCs. In addition, POSTN and FN1 were upregulated upon TGFβ stimulation in both (c) fetal and (d) adult EPDCs (data are representative of at least three independent EMT experiments). Figure S5. Migration and tube formation capacity of human EPDCs and sEPDCs. Scratch assays revealed that TGFβ stimulation decreased the migration rate of both (a) fetal and (b) adult sEPDCs. Furthermore, the dotted line, representing the migration rate of fetal and adult EPDCs, suggested that sEPDCs have an equal or even decreased ability to migrate. In the tube formation assay, comparing fetal and adult mesenchymal EPDCs, (c) adult sEPDCs had an increased ability to assemble into a closed tubular network. (d) Matrigel-based tube formation assays revealed that upon partial induction of EMT (untreated or TGFβ-treated) adult EPDCs formed more tubes. # p < 0.05. Figure S6. Fetal EPDCs are more prone to undergo EMT. (a) Upon removal of SB for a period of 4 days, WT1 protein was only withdrawn from the nucleus of fetal EPDCs. (b) Upon TGFβ stimulation, fetal EPDCs underwent EMT at a faster rate. This was shown by the occurrence of αSMA+ fibers in fetal cultures already after 24 hours (day 1) of stimulation (scale bar: 50 μm). (c) The epithelial-related factors, CDH1 and ANXA8, were increased in adult EPDCs, (d) while the mesenchymal genes TCF21 and VIM were upregulated in fetal EPDCs at baseline. # p < 0.05. (PDF 491 kb

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

<div><p>Aims</p><p>Hereditary 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.</p><p>Methods and results</p><p>After inducing myocardial infarction (MI), wild type (WT) and endoglin heterozygous (<i>Eng</i>^+/-) mice were treated for 5 days with the DPP4 inhibitor Diprotin A (DipA). DipA increased the number of CXCR4⁺ MNCs residing in the infarcted <i>Eng</i>^+/- hearts (<i>Eng</i>^+/- 73.17±12.67 vs. <i>Eng</i>^+/- treated 157.00±11.61, P = 0.0003) and significantly reduced infarct size (<i>Eng</i>^+/- 46.60±9.33% vs. <i>Eng</i>^+/- treated 27.02±3.04%, P = 0.03). Echocardiography demonstrated that DipA treatment slightly deteriorated heart function in <i>Eng</i>^+/- mice. An increased number of capillaries (<i>Eng</i>^+/- 61.63±1.43 vs. <i>Eng</i>^+/- treated 74.30±1.74, P = 0.001) were detected in the infarct border zone whereas the number of arteries was reduced (<i>Eng</i>^+/- 11.88±0.63 vs. <i>Eng</i>^+/- treated 6.38±0.97, P = 0.003). Interestingly, while less M2 regenerative macrophages were present in <i>Eng</i>^+/- hearts prior to DipA treatment, (WT 29.88±1.52% vs. <i>Eng</i>^+/- 12.34±1.64%, P<0.0001), DPP4 inhibition restored the number of M2 macrophages to wild type levels.</p><p>Conclusions</p><p>In this study, we demonstrate that systemic DPP4 inhibition restores the impaired MNC homing in <i>Eng</i>^+/- 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.</p></div