Preexisting Endothelial Cells Research Articles

Back and forth: History of and new insights on the vertebrate lymphatic valve.

Lymphatic valves develop from pre‐existing endothelial cells through a step‐wise process involving complex changes in cell shape and orientation, along with extracellular matrix interactions, to form two intraluminal leaflets. Once formed, valves prevent back‐flow within the lymphatic system to ensure drainage of interstitial fluid back into the circulatory system, thereby serving a critical role in maintaining fluid homeostasis. Despite the extensive anatomical characterization of lymphatic systems across numerous genus and species dating back several hundred years, valves were largely thought to be phylogenetically restricted to mammals. Accordingly, most insights into molecular and genetic mechanisms involved in lymphatic valve development have derived from mouse knockouts, as well as rare diseases in humans. However, we have recently used a combination of imaging and genetic analysis in the zebrafish to demonstrate that valves are a conserved feature of the teleost lymphatic system. Here, we provide a historical overview of comparative lymphatic valve anatomy together with recent efforts to define molecular pathways that contribute to lymphatic valve morphogenesis. Finally, we integrate our findings in zebrafish with previous work and highlight the benefits that this model provides for investigating lymphatic valve development.

Inhibiting Endothelial Cell Function in Normal and Tumor Angiogenesis Using BMP Type I Receptor Macrocyclic Kinase Inhibitors.

Simple SummaryAnti-angiogenesis agents have shown anti-cancer activity by preventing blood vessel ingrowth, thereby limiting tumour growth and metastasis. Although these molecules lead to prolonged overall survival of cancer patients, therapy resistance is easily acquired. Therefore, novel inhibitors against other signaling pathways mediating angiogenesis are needed to achieve more efficient and sustainable targeting of the angiogenesis process. Here, we synthesized and identified two compounds belonging to a new class of small molecules termed macrocyclics that selectively inhibit bone morphogenetic protein receptor kinase activity. One compound also inhibits vascular endothelial growth factor-induced signalling. Treatment studies using in vitro cultured cells and zebrafish embryos revealed that both compounds impaired endothelial cell function and decreased normal and tumour-induced angiogenesis. Both compounds might provide a steppingstone for the development of novel-angiogenesis therapeutic agents.Angiogenesis, i.e., the formation of new blood vessels from pre-existing endothelial cell (EC)-lined vessels, is critical for tissue development and also contributes to neovascularization-related diseases, such as cancer. Vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are among many secreted cytokines that regulate EC function. While several pharmacological anti-angiogenic agents have reached the clinic, further improvement is needed to increase clinical efficacy and to overcome acquired therapy resistance. More insights into the functional consequences of targeting specific pathways that modulate blood vessel formation may lead to new therapeutic approaches. Here, we synthesized and identified two macrocyclic small molecular compounds termed OD16 and OD29 that inhibit BMP type I receptor (BMPRI)-induced SMAD1/5 phosphorylation and downstream gene expression in ECs. Of note, OD16 and OD29 demonstrated higher specificity against BMPRI activin receptor-like kinase 1/2 (ALK1/2) than the commonly used small molecule BMPRI kinase inhibitor LDN-193189. OD29, but not OD16, also potently inhibited VEGF-induced extracellular regulated kinase MAP kinase phosphorylation in ECs. In vitro, OD16 and OD29 exerted strong inhibition of BMP9 and VEGF-induced ECs migration, invasion and cord formation. Using Tg (fli:EGFP) zebrafish embryos, we found that OD16 and OD29 potently antagonized dorsal longitudinal anastomotic vessel (DLAV), intra segmental vessel (ISV), and subintestinal vessel (SIV) formation during embryonic development. Moreover, the MDA-MB-231 breast cancer cell-induced tumor angiogenesis in zebrafish embryos was significantly decreased by OD16 and OD29. Both macrocyclic compounds might provide a steppingstone for the development of novel anti-angiogenesis therapeutic agents.

Angiogenesis after acute myocardial infarction.

Acute myocardial infarction (MI) inflicts massive injury to the coronary microcirculation leading to vascular disintegration and capillary rarefication in the infarct region. Tissue repair after MI involves a robust angiogenic response that commences in the infarct border zone and extends into the necrotic infarct core. Technological advances in several areas have provided novel mechanistic understanding of postinfarction angiogenesis and how it may be targeted to improve heart function after MI. Cell lineage tracing studies indicate that new capillary structures arise by sprouting angiogenesis from pre-existing endothelial cells (ECs) in the infarct border zone with no meaningful contribution from non-EC sources. Single-cell RNA sequencing shows that ECs in infarcted hearts may be grouped into clusters with distinct gene expression signatures, likely reflecting functionally distinct cell populations. EC-specific multicolour lineage tracing reveals that EC subsets clonally expand after MI. Expanding EC clones may arise from tissue-resident ECs with stem cell characteristics that have been identified in multiple organs including the heart. Tissue repair after MI involves interactions among multiple cell types which occur, to a large extent, through secreted proteins and their cognate receptors. While we are only beginning to understand the full complexity of this intercellular communication, macrophage and fibroblast populations have emerged as major drivers of the angiogenic response after MI. Animal data support the view that the endogenous angiogenic response after MI can be boosted to reduce scarring and adverse left ventricular remodelling. The improved mechanistic understanding of infarct angiogenesis therefore creates multiple therapeutic opportunities. During preclinical development, all proangiogenic strategies should be tested in animal models that replicate both cardiovascular risk factor(s) and the pharmacotherapy typically prescribed to patients with acute MI. Considering that the majority of patients nowadays do well after MI, clinical translation will require careful selection of patients in need of proangiogenic therapies.

No Evidence for Erythro-Myeloid Progenitor-Derived Vascular Endothelial Cells in Multiple Organs.

Endothelial cells are thought to emerge de novo from the mesoderm to form the entire circulatory system. Recently, erythro-myeloid progenitors (EMPs) have been proposed to be another remarkable developmental origin for blood vessels in multiple organs, including the hindbrain, liver, lung, and heart, as demonstrated by lineage tracing studies using different genetic tools. These observations challenge the current consensus that intraembryonic vessels are thought to expand solely by the proliferation of preexisting endothelial cells. Resolution of this controversy over the developmental origin of endothelial cells is crucial for developing future therapeutics for vessel-dependent organ repair and regeneration. To examine the contribution of EMPs to intraembryonic endothelial cells. We first used a transgenic mouse expressing a tamoxifen-inducible Mer-iCre fusion protein driven by the Csf1r (colony stimulating factor 1 receptor) promoter. Genetic lineage tracing based on Csf1r-Mer-iCre-Mer showed no contribution of EMPs to brain endothelial cells identified by several markers. We also generated a knock-in mouse line by inserting an internal ribosome entry site-iCre cassette into the 3' untranslated region of Csf1r gene to further investigate the cellular fates of EMPs. Similarly, we did not find any Csf1r-ires-iCre traced endothelial cells in brain, liver, lung, or heart in development either. Additionally, we found that Kit (KIT proto-oncogene receptor tyrosine kinase) was expressed not only in EMPs but also in embryonic hindbrain endothelial cells. Therefore, Kit promoter-driven recombinase, such as Kit-CreER, is a flawed tool for lineage tracing when examining the contribution of EMPs to hindbrain endothelial cells. We also traced CD45 (protein tyrosine phosphatase receptor type C; Ptprc)+ circulating EMPs and did not find any CD45 lineage-derived endothelial cells during development. Our study suggested that EMPs are not the origin of intraembryonic endothelial cells.

CYP4A/20-HETE regulates ischemia-induced neovascularization via its actions on endothelial progenitor and preexisting endothelial cells.

20-Hydroxyeicosatetraenoic acid (20-HETE) was recently identified as a novel contributor of ischemia-induced neovascularization based on the key observation that pharmacological interferences of CYP4A/20-HETE decrease ischemic neovascularization. The objective of the present study is to examine whether the underlying cellular mechanisms involve endothelial progenitor cells (EPCs) and preexisting endothelial cells (ECs). We found that ischemia leads to a time-dependent increase of cyp4a12 expression and 20-HETE production, which are endothelial in origin, using immunofluorescent microscopy, Western blot analysis, and LC-MS/MS. This is accompanied by increases in the tissue stromal cell-derived factor-1α (SDF-1α) expressions as well as SDF-1α plasma levels, EPC mobilization from bone marrow, and subsequent homing to ischemic tissues. Pharmacological interferences of CYP4A/20-HETE with a 20-HETE synthesis inhibitor, dibromo-dodecenyl-methylsulfimide (DDMS), or a 20-HETE antagonist, N-(20-hydroxyeicosa-6(Z), 15(Z)-dienoyl) glycine (6, 15-20-HEDGE), significantly attenuated these increases. Importantly, we also determined that 20-HETE plays a novel role in maintaining EPC functions and increasing the expression of Oct4, Sox2, and Nanog, which are indicative of increased progenitor cell stemness. Flow cytometric analysis revealed that pharmacological interferences of CYP4A/20-HETE decrease the EPC population in culture, whereas 20-HETE increases the cultured EPC population. Furthermore, ischemia also markedly increased the proliferation, oxidative stress, and ICAM-1 expression in the preexisting EC in the hindlimb gracilis muscles. We found that these increases were markedly negated by DDMS and 6, 15-20-HEDGE. Taken together, CYP4A/20-HETE regulates ischemia-induced compensatory neovascularization via its combined actions on promoting EPC and local preexisting EC responses that are associated with increased neovascularization. NEW & NOTEWORTHY CYP4A/20-hydroxyeicosatetraenoic acid (20-HETE) was recently discovered as a novel contributor of ischemia-induced neovascularization. However, the underlying molecular and cellular mechanisms are completely unknown. Here, we show that CYP4A/20-HETE regulates the ischemic neovascularization process via its combined actions on both endothelial progenitor cells (EPCs) and preexisting endothelial cells. Moreover, this is the first study, to the best of our knowledge, that associates CYP4A/20-HETE with EPC differentiation and stemness.

The CYP4A/20‐HETE Axis Regulates Ischemia‐induced Neovascularization via Its Actions on Endothelial Progenitor and Preexisting Endothelial Cells

20‐hydroxyeicosatetraenoic acid (20‐HETE) was recently identified as a novel contributor of ischemia‐induced neovascularization based on the key observation that pharmacological interferences of the CYP4A/20‐HETE axis decrease ischemic neovascularization. The objective of the present study is to determine whether the underlying cellular mechanisms involve endothelial progenitor cells (EPC) and pre‐existing endothelial cells (EC).We found that ischemia leads to a time‐dependent increase in 20‐HETE that is endothelial in origin using LC/MS/MS analysis and immunofluorescent (IF) microscopy. This is accompanied by increases in SDF‐1a expressions, EPC mobilization from bone marrow, and subsequent homing to ischemic tissues. Pharmacological interferences of the CYP4A/20‐HETE axis with a 20‐HETE synthesis inhibitor Dibromo‐dodecenyl‐methylsulfimide(DDMS) or a 20‐HETE antagonist N‐(20‐hydroxyeicosa‐6(Z), 15(Z)‐dienoyl) glycine(6, 15‐20‐HEDGE) significantly attenuated these increases. Importantly, we also determined that 20‐HETE plays a novel role in maintaining EPC functions and increases in the expression of Oct4, Sox2, and Nanog expression that are indicative of increased progenitor cell stemness. Flow cytometric analysis revealed that pharmacological interferences of the CYP4A/20‐HETE axis decrease the EPC population in culture, whereas 20‐HETE increases the cultured EPC population. Furthermore, ischemia also markedly increased the proliferation, oxidative stress, and ICAM‐1 expression in preexisting EC in the hindlimb gracilis muscles. We found that these increases were markedly negated by DDMS and 6, 15‐20‐HEDGE.Taken together, the CYP4A/20‐HETE axis regulates ischemia‐induced compensatory neovascularization via its combined actions on promoting EPC and local preexisting EC responses that are associated with increased neovascularization.Support or Funding InformationAmerican Heart Association [Grant 11SDG6870004]; National Institutes of Health [Grants HL34300], [Grant DK38226]; Robert A. Welch Foundation [GL625910]This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vasculogenic Stem and Progenitor Cells in Human: Future Cell Therapy Product or Liquid Biopsy for Vascular Disease.

New blood vessel formation in adults was considered to result exclusively from sprouting of preexisting endothelial cells, a process referred to angiogenesis. Vasculogenesis, the formation of new blood vessels from endothelial progenitor cells, was thought to occur only during embryonic life. Discovery of adult endothelial progenitor cells (EPCs) in 1997 opened the door for cell therapy in vascular disease. Endothelial progenitor cells contribute to vascular repair and are now well established as postnatal vasculogenic cells in humans. It is now admitted that endothelial colony-forming cells (ECFCs) are the vasculogenic subtype. ECFCs could be used as a cell therapy product and also as a liquid biopsy in several vascular diseases or as vector for gene therapy. However, despite a huge interest in these cells, their tissue and molecular origin is still unclear. We recently proposed that endothelial progenitor could come from very small embryonic-like stem cells (VSELs) isolated in human from CD133 positive cells. VSELs are small dormant stem cells related to migratory primordial germ cells. They have been described in bone marrow and other organs. This chapter discusses the reported findings from in vitro data and also preclinical studies that aimed to explore stem cells at the origin of vasculogenesis in human and then explore the potential use of ECFCs to promote newly formed vessels or serve as liquid biopsy to understand vascular pathophysiology and in particular pulmonary disease and haemostasis disorders.

The Development and Regeneration of Coronary Arteries.

In this review, we aim to summarize and discuss the cellular origins of the coronary endothelial cells during development and neovascularization in the adult stage after cardiac injury. Recent work identified three different developmental origins for coronary endothelial cells: proepicardium, endocardium, and sinus venosus. However, the level of contribution by each source remains debated. During heart injury and regeneration, although multiple cell types such as endothelial progenitor cells, epicardial cells, and endocardial cells were reported to contribute neovascularization, convincing evidence is still lacking.. Recently, fibroblasts were reported to contribute to endothelial cells after cardiac injury through mesenchymal-to-endothelial transition. A subsequent study demonstrated that pre-existing endothelial cells mainly mediate cardiac neovascularization after injury. The developmental origins of coronary vessels are diverse and further studies are needed to address the exact contribution from each source and the molecular mechanism governing distinct vessel formation programs. In the adult stage, neovascularization is mainly mediated by the pre-existing endothelial cells, with negligible contribution from other sources.

Basic research: Where do new endothelial cells come from in the injured heart?

Revascularization of ischaemia-injured myocardium is critical for functional recovery. A new study shows that endothelial cells of neovessels in the injured heart derive from pre-existing endothelial cells. This new finding focuses research on therapeutic strategies to direct the neovasculature to deliver oxygen and nutrients effectively to the ischaemic myocardium.

Preexisting endothelial cells mediate cardiac neovascularization after injury.

The mechanisms that promote the generation of new coronary vasculature during cardiac homeostasis and after injury remain a fundamental and clinically important area of study in the cardiovascular field. Recently, it was reported that mesenchymal-to-endothelial transition (MEndoT) contributes to substantial numbers of coronary endothelial cells after myocardial infarction. Therefore, the MEndoT has been proposed as a paradigm mediating neovascularization and is considered a promising therapeutic target in cardiac regeneration. Here, we show that preexisting endothelial cells mainly beget new coronary vessels in the adult mouse heart, with essentially no contribution from other cell sources through cell-lineage transdifferentiation. Genetic-lineage tracing revealed that cardiac fibroblasts expand substantially after injury, but do not contribute to the formation of new coronary blood vessels, indicating no contribution of MEndoT to neovascularization. Moreover, genetic-lineage tracing with a pulse-chase labeling strategy also showed that essentially all new coronary vessels in the injured heart are derived from preexisting endothelial cells, but not from other cell lineages. These data indicate that therapeutic strategies for inducing neovascularization should not be based on targeting presumptive lineage transdifferentiation such as MEndoT. Instead, preexisting endothelial cells appear more likely to be the therapeutic target for promoting neovascularization and driving heart regeneration after injury.

Tauroursodeoxycholic acid, a bile acid, promotes blood vessel repair by recruiting vasculogenic progenitor cells.

Although serum bile acid concentrations are approximately 10 µM in healthy subjects, the crosstalk between the biliary system and vascular repair has never been investigated. In this study, tauroursodeoxycholic acid (TUDCA) induced dissociation of CD34(+) hematopoietic stem cells (HSCs) from stromal cells by reducing adhesion molecule expression. TUDCA increased CD34(+) /Sca1(+) progenitors in mice peripheral blood (PB), and CD34(+) , CD31(+) , and c-kit(+) progenitors in human PB. In addition, TUDCA increased differentiation of CD34(+) HSCs into EPC lineage cells via Akt activation. EPC invasion was increased by TUDCA, which was mediated by fibroblast activating protein via Akt activation. Interestingly, TUDCA induced integration of EPCs into human aortic endothelial cells (HAECs) by increasing adhesion molecule expression. In the mouse hind limb ischemia model, TUDCA promoted blood perfusion by enhancing angiogenesis through recruitment of Flk-1(+) /CD34(+) and Sca-1(+) /c-kit(+) progenitors into damaged tissue. In GFP(+) bone marrow-transplanted hind limb ischemia, TUDCA induced recruitment of GFP(+) /c-kit(+) progenitors to the ischemic area, resulting in an increased blood perfusion ratio. Histological analysis suggested that GFP(+) progenitors mobilized from bone marrow, integrated into blood vessels, and differentiated into VEGFR(+) cells. In addition, TUDCA decreased cellular senescence by reducing levels of p53, p21, and reactive oxygen species and increased nitric oxide. Transplantation of TUDCA-primed senescent EPCs in hind limb ischemia significantly improved blood vessel regeneration, as compared with senescent EPCs. Our results suggested that TUDCA promoted neovascularization by enhancing the mobilization of stem/progenitor cells from bone marrow, their differentiation into EPCs, and their integration with preexisting endothelial cells.

Abstract 1004: LHT7, a heparin-taurocholate conjugate, inhibits multiple stages of angiogenesis by targeting VEGF, FGF2 and PDGF-B

Abstract Angiogenesis is a critical process in tumor progression and metastasis. Therefore, many angiogenesis inhibitors have been developed as cancer therapeutics, especially targeting vascular endothelial growth factor (VEGF) signaling pathway. These VEGF blockers effectively inhibited the tumor growth initially, but failed to produce enduring clinical response due to occurrence of acquired resistance. Mechanism of the resistance is not clearly understood so far. However, many studies have reported an upregulation of alternative angiogenic pathways when VEGF was blocked that led to recurrence of tumor angiogenesis. Other studies suggested that stabilization of endothelium by mural cells is also responsible for the resistance. Therefore, simultaneous inhibition of the several angiogenic pathways that are involved in multiple stages of angiogenesis would potentially overcome this limitation. To develop such an angiogenesis inhibitor, we regarded heparin as a lead compound, since wide-array of pro-angiogenic factors are heparin-binding proteins. We have previously reported a heparin-derived angiogenesis inhibitor, LHT7, which blocks VEGF signaling pathway and inhibit tumor angiogenesis. In this study, we show that LHT7 could block fibroblast growth factor 2 (FGF2) and platelet-derived growth factor B (PDGF-B) signaling pathway as well as VEGF. We prepared a set of in vitro studies that represent each step of the initial robust angiogenesis, including endothelial cell migration, angiogenic sprouting from pre-existing vasculatures and endothelial cell differentiation. An in vitro experiment that mimic the maturation of endothelium was also carried out. The inhibitory effect of LHT7 on VEGF, FGF2 and PDGF-B at the molecular level was studied by determining the phosphotyrosine level of the corresponding receptors. The effect was further assessed in vivo by using matrigel plug assay. All the results indicated that LHT7 blocked VEGF, FGF2 and PDGF-B, leading to inhibition of multiple stages of angiogenic process in terms of initial angiogenic response and maturation of endothelium. In addition, numbers of studies have reported vascular normalization during treatment of VEGF blockers, which is closely related to vascular maturation. However, dynamic contrast-enhanced magnetic resonance imaging of LHT7-treated tumor-bearing animals did not show any sign of vascular normalization and directly lead to decreased perfusion of blood throughout the tumor. Our results show that LHT7 could effectively inhibit tumor angiogenesis by blocking multiple stages of angiogenesis and potentially overcome the acquired resistance against angiogenesis inhibitors. Furthermore, they imply that heparin could be an excellent lead compound to develop a multi-targeted angiogenesis inhibitor. Citation Format: Seung Woo Chung, Jeong Uk Choi, Sang Yoon Kim, Youngro Byun. LHT7, a heparin-taurocholate conjugate, inhibits multiple stages of angiogenesis by targeting VEGF, FGF2 and PDGF-B. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1004. doi:10.1158/1538-7445.AM2014-1004

Prostate cancer and new insights in angiogenesis.

Prostate cancer and new insights in angiogenesis.

EComment. Potential therapeutic agents in vasoplegic syndrome after cardiac surgery

Vasoplegic syndrome (VS) or vasodilatory shock frequently occurs after cardiac surgery, particularly with cardiopulmonary bypass(CPB). It is often associated with high morbidity and mortality, especially if it is resistant to intravascular volume expansion and conventional vasoconstrictive drugs [1]. This complication is similar to septic shock and characterized by severe and persistent hypotension, decreased systemic vascular resistance (SVR), normal or increased cardiac output (CO), low intra-cardiac filling pressures, and the necessity for high-dose catecholamines. Until today, the aetiology of VS is not fully understood. However, this syndrome is thought to be caused by the inflammatory-mediated dysregulation of endothelium homeostasis and subsequent endothelial dysfunction, including endothelial cell activation, endothelial permeability augmentation, coagulation defects, production of different regulators, and vascular tone disorders [1]. We read with great interest the paper by Kortekaas et al [2]. The authors report that pre-existing endothelial cell activation, reflected by higher baseline von Willebrand factor pro-peptide and soluble P-selectin levels is a predisposing factor for VS after mitral valve surgery. They also conclude that this activation may have resulted in desensibilization of endothelium in patients with VS. We would like to add some comments on possible agents for the management of VS VS is attributed to a combination of endothelial injury, arginine-vasopressin system dysfunction, and the release of vasodilatory inflammatory mediators[1]. Although these markers may be useful for the identification of patients at increased risk of developing VS, effective treatment strategies for this syndrome are currently lacking. The choice of vasoconstrictive agents for VS is a matter of controversy. To maintain adequate haemodynamics for VS, the conventional treatment based on fluid resuscitation and catecholamine drugs is ineffective in a subset of patients with profound vasodilatation[1]. Arginine vasopressin (AVP) and methylene blue (MB) may be used as additional rescue or a preventative treatment for VS [1, 3]. AVP is well-known to treat norepinephrine-resistant vasodilatation but may be associated rebound hypotension after discontinuation [3,4]. AVP significantly increases mean arterial pressure (MAP) and SVR, and results in a marked decrease in norepinephrine doses [1]. Tayama and associates [3] treated postoperative refractory low SVR hypotension concomitant with pulmonary hypertension by AVP. They reported that exogenous AVP normalized SVR and increased MAP. They also observed that AVP enhanced urine output and improved respiratory function. Noto and co-workers [4] reported that they treated six patients with terlipressin (TP; 1 mg bolus), a synthetic analogue of AVP for post-CPB refractory vasodilatory hypotension. In these patients, the authors suggested that exogenous TP administration normalized SVR and increased MAP with a minimum effect on pulmonary pressure. MB, an inhibitor of nitric oxide synthase and guanylate cyclase, seems to be a possible effective therapeutical option and to improve outcomes in patients with catecholamine-resistant VS after cardiac surgery [1, 5]. In a prospective observational study, Leyh and colleagues [5] tested the effect of MB (2 mg/kg over 20 minutes) in 54 patients with norepinephrine-resistant VS. In this study, MAP increased and norepinephrine dose decreased 1 hour after MB administration. These changes were accompanied by a significant decrease in CO. Finally, we think that further studies are necessary to find the most appropriate possible therapeutic agents for VS. Conflict of interest: none declared

Pre-existing endothelial cell activation predicts vasoplegia after mitral valve surgery†

Post-cardiac surgery vasoplegia is a common complication of cardiac surgery, characterized by profound loss of systemic vascular resistance. This results in severe hypotension, high cardiac output and metabolic acidosis reflecting inadequate tissue perfusion. The pathophysiological mechanisms underlying this syndrome remain unknown. We hypothesized that this vasoplegia reflects endothelial dysfunction, either as pre-existing condition or as a consequence of the surgical procedure. To examine these mechanisms, six established and distinct markers of endothelial cell activation were measured pre- and perioperatively in patients undergoing mitral valve surgery. Arterial (radial artery) and myocardial venous blood samples (coronary sinus) were collected simultaneously over the reperfused heart at various time points during the first hour after reperfusion. Additional samples were collected at baseline (brachial vein) and 1 day post-reperfusion (radial artery). Post-cardiac surgery vasoplegia was defined as a mean arterial blood pressure of <60 mmHg, with a cardiac index of ≥2.2 l/min/m(2) treated with continuous intravenous administration of norepinephrine. No myocardial release of endothelial cell activation markers was observed upon reperfusion in patients with vasoplegia (n = 15; mean age 71 years, 73% male). In contrast, in patients without vasoplegia (n = 24; mean age 64 years, 54% male), reperfusion was characterized by a myocardial release of three endothelial cell activation markers. Myocardial von Willebrand Factor propeptide, osteoprotegerin and interleukin-8 were increased 107% (P < 0.001), 106% (P = 0.02) and 116% (P = 0.009), respectively, compared with arterial levels upon reperfusion. Similar systemic levels of all markers were found upon reperfusion in both groups, except for 120% increased soluble P-selectin (sP-selectin) levels in vasoplegia patients (P = 0.03). Remarkably, postoperative vasoplegia was identified with baseline von Willebrand Factor propeptide levels with a cut-off value of 11.9 nM as well as with baseline sP-selectin levels with a cut-off value of 64.4 ng/ml. Pre-existing endothelial cell activation, reflected by higher baseline von Willebrand Factor propeptide and sP-selectin levels, is a predisposing factor for post-cardiac surgery vasoplegia. The pre-existing endothelial cell activation may have resulted in desensibilization of endothelium in patients who develop vasoplegic syndrome, resulting in no myocardial release of endothelial cell activation markers upon reperfusion.

Gene expression analysis reveals an angiogenic profile in uterine leiomyoma pseudocapsule

The pseudocapsule (PC) of the uterine leiomyoma (UL) is an anatomic entity that surrounds the myoma separating it from the myometrium (UM). Although a number of microarray experiments have identified differences in gene expression profile in the UL when compared with the UM, there is a lack of systematic studies on the PC. In this study, quantitative RT-PCR analysis was performed on 18 matched PC, UL and UM specimens and results showed that the PC displays a specific gene expression profile. The low expression level of insulin-like growth factor (IGF-2), a fibroid specific marker, that we found in the PC and the UM when compared with the UL, clearly indicates that the PC is in structural continuity with the UM. However, the significant increase in endoglin expression level in PC with respect to the UL and UM indicates that an active neoangiogenesis is present in PC. Conversely, other angiogenic factors such as von Willebrand factor (vWF) and vascular endothelial growth factor A (VEGF-A) seem to have little influence on the PC angiogenesis. Because the endoglin is preferentially expressed in proliferating endothelial cells, whereas the vWF and VEGF-A are preferentially expressed in preexisting endothelial cells, our idea is that the angiogenic activity in the PC is linked to wound healing. The angiogenic activity is also sustained by intermediate expression level of cystein-rich angiogenesis inducer 61, connective tissue growth factor and collagen 4α2 genes all involved in the neoangiogenesis, that we detected in the PC. Taken together our data demonstrate that the specific expression pattern observed in the PC could be the response of the uterine wall's smooth cells to the tension imposed by the tumor. As a consequence, a neovascular structure is generated involving regenerative processes. For these reasons, we suggest that the laparoscopic intracapsular myomectomy (LIM), a new surgical technique that preserves the PC during the UL removal, should always be preferred, to favor a faster and proper uterine healing.

Plaque neovascularization: defense mechanisms, betrayal, or a war in progress

Angiogenesis is induced from sprouting of preexisting endothelial cells leading to neovascularization. Imbalance in the angiogenic and antiangiogenic mediators triggers angiogenesis, which may be physiological in the normal state or pathological in malignancy and atherosclerosis. Physiologic angiogenesis is instrumental for restoration of vessel wall normoxia and resolution inflammation, leading to atherosclerosis regression. However, pathological angiogenesis enhances disease progression, increasing macrophage infiltration and vessel wall thickness, perpetuating hypoxia and necrosis. In addition, thin-walled fragile neovessels may rupture, leading to intraplaque hemorrhage. Lipid-rich red blood cell membranes and free hemoglobin are detrimental to plaque composition, increasing inflammation, lipid core expansion, and oxidative stress. In addition, associated risk factors that include polymorphysms in the haptoglobin genotype and diabetes mellitus may modulate the features of plaque vulnerability. This review will focus on physiological and pathological angiogenesis in atherosclerosis and summarizes the current status of anti-vascular endothelial growth factor (VEGF) therapy, microvascular rarefaction, and possible statin-mediated effects in atherosclerosis neovascularization.

Process of Coronary Capillary Tube Formation by Mononuclear Cells Expressing β-Actin in the Ischemic Myocardium

Background: Capillary tube formation is essential for angiogenesis. It was previously thought that capillary tubes are formed through self-division of pre-existing endothelial cells (ECs) or folding of EC-progenitor cells. We found that circulating mononuclear cells that express β-actin (β-MNCs), participate in arteriogenesis, (formation of arterioles and arteries, and their growth) but it remained obscure whether and how they participate in capillary tube formation. Methods: We induced acute myocardial infarction in beagles, and examined the process of capillary tube formation by β-MNCs in the infarcted myocardium. Results: β-MNCs were recruited to coronary vessels, in particular to capillaries, in the border zone of infarcted myocardium. β-MNC nuclei were either round or flat, indicating existence of several species of β-MNCs. They sprouted out from a pre-existing coronary arteriole, capillary or venule into the interstitial space, and adhered to each other in tandem. An intracellular cavity was formed by the loss of granular cytoplasm in each cell. The intercellular junctions which connected cell-to-cell disappeared, and the cavities connected with each other to form a capillary tube. New β-MNCs moved to the distal end of the tube and migrated outside to extend the capillary tube. ECs of the capillary tubes thus formed possessed CD31, a marker for EC, but not β-actin. Their nuclei were either round or flat, indicating existence of two different capillary tubes. Conclusions: Coronary capillary tube formation in the ischemic myocardium is initiated by β-MNCs, not by their folding but by intracellular cavity formation through the loss of their granular cytoplasm and consequent tube formation by cavity-to-cavity connection.

Cilostazol Activates Function of Bone Marrow-Derived Endothelial Progenitor Cell for Re-endothelialization in a Carotid Balloon Injury Model

BackgroundCilostazol(CLZ) has been used as a vasodilating anti-platelet drug clinically and demonstrated to inhibit proliferation of smooth muscle cells and effect on endothelial cells. However, the effect of CLZ on re-endothelialization including bone marrow (BM)-derived endothelial progenitor cell (EPC) contribution is unclear. We have investigated the hypothesis that CLZ might accelerate re-endothelialization with EPCs.Methodology/Principal FindingsBalloon carotid denudation was performed in male Sprague-Dawley rats. CLZ group was given CLZ mixed feed from 2weeks before carotid injury. Control group was fed normal diet. CLZ accelerated re-endothelialization at 2 weeks after surgery and resulted in a significant reduction of neointima formation 4 weeks after surgery compared with that in control group. CLZ also increased the number of circulating EPCs throughout the time course. We examined the contribution of BM-derived EPCs to re-endothelialization by BM transplantation from Tie2/lacZ mice to nude rats. The number of Tie2-regulated X-gal positive cells on injured arterial luminal surface was increased at 2 weeks after surgery in CLZ group compared with that in control group. In vitro, CLZ enhanced proliferation, adhesion and migration activity, and differentiation with mRNA upregulation of adhesion molecule integrin αvβ3, chemokine receptor CXCR4 and growth factor VEGF assessed by real-time RT-PCR in rat BM-derived cultured EPCs. In addition, CLZ markedly increased the expression of SDF-1α that is a ligand of CXCR4 receptor in EPCs, in the media following vascular injury.Conclusions/SignificanceCLZ promotes EPC mobilization from BM and EPC recruitment to sites of arterial injury, and thereby inhibited neointima formation with acceleration of re-endothelialization with EPCs as well as pre-existing endothelial cells in a rat carotid balloon injury model. CLZ could be not only an anti-platelet agent but also a promising tool for endothelial regeneration, which is a key event for preventing atherosclerosis or restenosis after vascular intervention.

The role of fibrin E on the modulation of endothelial progenitors adhesion, differentiation and angiogenic growth factor production and the promotion of wound healing

Severe skin loss constitutes a major unsolved clinical problem worldwide. For this reason, in the last decades there has been a major push towards the development of novel therapeutic approaches to enhance skin wound healing. Neo-vessel formation through angiogenesis is a critical step during the wound healing process. Besides the contribution of pre-existing endothelial cells (EC), endothelial progenitor cells (EPCs) have also been implicated in wound healing acting either by differentiating into EC that incorporate the neo-vessels, or via the production of paracrine factors that improve angiogenesis. Here we tested the importance of different extracellular matrices (ECM) in regulating the angiogenic and wound healing potential of cord blood-derived EPC (CB-EPC). We compared the properties of several ECM and particularly of fibrin fragment E (FbnE) in regulating EPC adhesion, proliferation, differentiation and healing-promotion in vitro and in vivo. Our results show that CB-EPCs have increased adhesion and endothelial differentiation when plated on FbnE compared to collagens, fibronectin or fibrin. Using integrin neutralizing antibodies, we show that CB-EPC adhesion to FbnE is mediated by integrin α5β1. Gene expression analysis of CB-EPCs plated on different substrates revealed that CB-EPC grown on FbnE shows increased expression of paracrine factors such as VEGF-A, TGF-β1, SDF-1, IL-8 and MIP-1α. Accordingly, conditioned media from CB-EPC grown on FbnE induced EC tube formation and monocyte migration in vitro. To test the wound healing effects of FbnE in vivo we used an FbnE enriched scaffold in a cutaneous wound healing mouse model. In accordance with our in vitro data, co-administration of the FbnE enriched scaffold with CB-EPC significantly accelerated wound closure and wound vascularization, compared FbnE enriched scaffold alone or to using collagen-based scaffolds. Our results show that FbnE modulates several CB-EPC properties in vivo and in vitro, and as such promotes wound healing. We suggest the use of FbnE-based scaffolds represents a promising approach to resolve wound healing complications arising from different pathologies.