Adaptive Angiogenesis Research Articles

Development of novel technology for the visualization and quantitation of microvascular progenitor-driven adaptive angiogenesis during chronic lung disease

Introduction: Adaptive angiogenesis can drive repair or underlie the pathogenesis of tissue remodeling. Pulmonary vascular dysfunction is a major manifestation of chronic lung disease (CLD) but the role of angiogenesis in CLD is not well defined. Microvascular capillaries are the vessels most affected by pruning and remodeling in the lung resulting in reduced capillary length and density. Our lab has previously demonstrated that microvascular progenitors (ABCG2+ MVPCs) drive adaptive angiogenesis or loss of tissue structure downstream of the Wnt or mTOR signaling pathways. Therefore, we hypothesize that visualization of the pulmonary microvasculature and MVPC-driven adaptive angiogenesis in three-dimensions is essential to define the role of MVPC-driven adaptive angiogenesis during repair or the pathogenesis of CLD. Methods: To evaluate the contribution of Wnt activated MVPCs to angiogenesis in three-dimensions during injury repair, we crossed ABCG2-CreERT2 mice to a BOE strain (flΔex3) with stabilized β-catenin expression and a fluorescent membrane eGFP reporter strain to facilitate lineage tracing. The development and resolution of fibrosis were induced in vivo via aspiration of bleomycin sulfate (0.05U). Mice were retro-orbitally perfused with fluorescently conjugated tomato lectin. Lung tissue was inflated with agarose and up to 500μm thickness precision cut lung slices (PCLS) were mounted for imaging. PCLS were imaged on the Zeiss LSM900 and Z-stacks were obtained. Imaris software (Oxford Instruments) was used for three-dimensional reconstruction and FIJI and Wimasis Image Analysis (Onimagin Technologies) were utilized to segment vessels, quantitate vessel density, volume, diameter, and length. Results: Following perfusion, vascular staining of both the lung and kidney was observed, suggesting successful systemic perfusion and labeling. The pulmonary microvasculature was visualized to a diameter of 1-3μm in high fidelity. We observed a significant reduction in vessel density in mice treated with bleomycin compared to mice treated with a vehicle control. In our three-dimensional reconstructions we observed increased density of ABCG2+ MVPC-derived de novo angiogenic structures in areas of fibrosis. Conclusion: De novo angiogenesis driven by ABCG2+ MVPCs likely plays a role in tissue repair following bleomycin induced lung injury. In addition, we have developed a simple and reliable perfusion technique allowing for the visualization and quantitation of the pulmonary microvasculature in three-dimensions. This tool will allow the quantitation of microvessel remodeling underlying pathogenesis and repair in multiple lung injury models. Thus, this methodology will further support the study and understanding of angiogenesis during CLD and repair following injury. This approach may be utilized in various organ systems to visualize microvasculature structure and its role in disease. NIA RAG073317 NHLBI 1R35HL161238. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exercise adaptations in COPD: the pulmonary perspective.

In chronic obstructive pulmonary disease (COPD), the progressive loss of lung tissue is widely considered irreversible. Thus, various treatment and rehabilitation schemes, including exercise-based pulmonary rehabilitation (PR) are thought to slow down but not reverse or halt the disease. Nonetheless, the adult lung conceals the intrinsic capacity for de novo lung tissue formation in the form of abundant progenitor/stem cell populations. In COPD, these maintain their differentiation potential but appear to be halted by a state of cellular senescence in the mesenchyme, which normally functions to support and coordinate their function. We propose that notably high-intensity interval training may improve pulmonary gas exchange during exercise in patients with COPD by interrupting mesenchymal senescence, thus reestablishing adaptive angiogenesis. By means of this, the downward spiral of dyspnea, poor quality of life, physical inactivity, and early death often observed in COPD may be interrupted. If this is the case, the perception of the regenerative capacity of the lungs will be fundamentally changed, which will warrant future clinical trials on various exercise schemes and other treatments targeting the formation of new lung tissue in COPD.

GDF15 and Cardiac Cells: Current Concepts and New Insights.

Growth and differentiation factor 15 (GDF15) belongs to the transforming growth factor-β (TGF-β) superfamily of proteins. Glial-derived neurotrophic factor (GDNF) family receptor α-like (GFRAL) is an endogenous receptor for GDF15 detected selectively in the brain. GDF15 is not normally expressed in the tissue but is prominently induced by “injury”. Serum levels of GDF15 are also increased by aging and in response to cellular stress and mitochondrial dysfunction. It acts as an inflammatory marker and plays a role in the pathogenesis of cardiovascular diseases, metabolic disorders, and neurodegenerative processes. Identified as a new heart-derived endocrine hormone that regulates body growth, GDF15 has a local cardioprotective role, presumably due to its autocrine/paracrine properties: antioxidative, anti-inflammatory, antiapoptotic. GDF15 expression is highly induced in cardiomyocytes after ischemia/reperfusion and in the heart within hours after myocardial infarction (MI). Recent studies show associations between GDF15, inflammation, and cardiac fibrosis during heart failure and MI. However, the reason for this increase in GDF15 production has not been clearly identified. Experimental and clinical studies support the potential use of GDF15 as a novel therapeutic target (1) by modulating metabolic activity and (2) promoting an adaptive angiogenesis and cardiac regenerative process during cardiovascular diseases. In this review, we comment on new aspects of the biology of GDF15 as a cardiac hormone and show that GDF15 may be a predictive biomarker of adverse cardiac events.

Filamin A Regulates Cardiovascular Remodeling.

Filamin A (FLNA) is a large actin-binding cytoskeletal protein that is important for cell motility by stabilizing actin networks and integrating them with cell membranes. Interestingly, a C-terminal fragment of FLNA can be cleaved off by calpain to stimulate adaptive angiogenesis by transporting multiple transcription factors into the nucleus. Recently, increasing evidence suggests that FLNA participates in the pathogenesis of cardiovascular and respiratory diseases, in which the interaction of FLNA with transcription factors and/or cell signaling molecules dictate the function of vascular cells. Localized FLNA mutations associate with cardiovascular malformations in humans. A lack of FLNA in experimental animal models disrupts cell migration during embryogenesis and causes anomalies, including heart and vessels, similar to human malformations. More recently, it was shown that FLNA mediates the progression of myocardial infarction and atherosclerosis. Thus, these latest findings identify FLNA as an important novel mediator of cardiovascular development and remodeling, and thus a potential target for therapy. In this update, we summarized the literature on filamin biology with regard to cardiovascular cell function.

Resident mesenchymal vascular progenitors modulate adaptive angiogenesis and pulmonary remodeling via regulation of canonical Wnt signaling.

Adaptive angiogenesis is necessary for tissue repair, however, it may also be associated with the exacerbation of injury and development of chronic disease. In these studies, we demonstrate that lung mesenchymal vascular progenitor cells (MVPC) modulate adaptive angiogenesis via lineage trace, depletion of MVPC, and modulation of β‐catenin expression. Single cell sequencing confirmed MVPC as multipotential vascular progenitors, thus, genetic depletion resulted in alveolar simplification with reduced adaptive angiogenesis. Following vascular endothelial injury, Wnt activation in MVPC was sufficient to elicit an emphysema‐like phenotype characterized by increased MLI, fibrosis, and MVPC driven adaptive angiogenesis. Lastly, activation of Wnt/β‐catenin signaling skewed the profile of human and murine MVPC toward an adaptive phenotype. These data suggest that lung MVPC drive angiogenesis in response to injury and regulate the microvascular niche as well as subsequent distal lung tissue architecture via Wnt signaling.

Disruption of lineage specification in adult pulmonary mesenchymal progenitor cells promotes microvascular dysfunction.

Pulmonary vascular disease is characterized by remodeling and loss of microvessels and is typically attributed to pathological responses in vascular endothelium or abnormal smooth muscle cell phenotypes. We have challenged this understanding by defining an adult pulmonary mesenchymal progenitor cell (MPC) that regulates both microvascular function and angiogenesis. The current understanding of adult MPCs and their roles in homeostasis versus disease has been limited by a lack of genetic markers with which to lineage label multipotent mesenchyme and trace the differentiation of these MPCs into vascular lineages. Here, we have shown that lineage-labeled lung MPCs expressing the ATP-binding cassette protein ABCG2 (ABCG2+) are pericyte progenitors that participate in microvascular homeostasis as well as adaptive angiogenesis. Activation of Wnt/β-catenin signaling, either autonomously or downstream of decreased BMP receptor signaling, enhanced ABCG2+ MPC proliferation but suppressed MPC differentiation into a functional pericyte lineage. Thus, enhanced Wnt/β-catenin signaling in ABCG2+ MPCs drives a phenotype of persistent microvascular dysfunction, abnormal angiogenesis, and subsequent exacerbation of bleomycin-induced fibrosis. ABCG2+ MPCs may, therefore, account in part for the aberrant microvessel function and remodeling that are associated with chronic lung diseases.

Syndecan-4 deficiency accelerates the transition from compensated hypertrophy to heart failure following pressure overload.

Increasing evidence suggests that a mismatch between angiogenesis and myocardial growth contributes to the transition from adaptive cardiac hypertrophy to heart failure following pressure overload. Syndecan-4 is a transmembrane proteoglycan that binds to growth factors and extracellular matrix proteins and is critical in focal adhesion formation. However, its effects on coronary angiogenesis during pressure overload-induced heart failure have not been studied. Here, we hypothesize that syndecan-4 modulates cardiac remodeling in response to pressure overload through its ability to regulate adaptive angiogenesis. Syndecan-4 knockout (syndecan-4 KO) and wild-type (WT) mice were subjected to pressure overload induced by transverse aortic constriction (TAC). Syndecan-4 KO mice exhibited reduced capillary density, attenuated cardiomyocyte size, and worsened left ventricular cardiac function after TAC surgery compared with WT mice. Moreover, syndecan-4 KO mice showed a significant decrease in protein kinase C alpha expression. Our data suggest that syndecan-4 is essential for the compensated hypertrophy and the maintenance of cardiac function during the process of heart failure following pressure overload.

Endostatin a Potential Biomarker for Heart Failure with Preserved Ejection Fraction.

BackgroundEndostatin is a circulating endogenous angiogenesis inhibitor preventing neovascularization. Previous studies demonstrated the prognostic value of Endostatin among patients with heart failure with reduced ejection fraction (HFrEF). However, the role of Endostatin among patients with heart failure with preserved ejection fraction (HFpEF) remains unclear.ObjectiveThis study aimed to investigate the association between serum Endostatin levels, natriuretic peptide levels and the severity of left ventricular diastolic dysfunction and the diagnosis of HFpEF.MethodsEndostatin serum concentrations were measured in 301 patients comprising 77 HFpEF patients, 169 patients with asymptomatic left ventricular diastolic dysfunction (ALVDD), and 55 controls with normal cardiac function.ResultsEndostatin serum levels were significantly elevated in patients with HFpEF (median/interquartile range 179.0 [159-220]) and ALVDD (163.8 [145.4-191.3]) compared to controls (149.1 [130.6-176.9]), p < 0.001 and p = 0.004, respectively) and significant correlated with N-terminal pro B-type natriuretic peptide (NT-proBNP).ConclusionsThis hypothesis-generating pilot study gives first evidence that Endostatin correlates with the severity of diastolic dysfunction and may become a novel biomarker for HFpEF. We hypothesize a rise in Endostatin levels may reflect inhibition of adaptive angiogenesis and adverse cardiac remodeling.

Changes in VEGF and bFGF serum concentration after long-term sports training in young athletes – The significance of adaptive angiogenesis in arterial blood pressure adjustment

PurposeThe aim of this investigation was to assess the influence of an 8-month period of physical training on serum concentration of a number of angiogenic growth factors and resting blood pressure in adolescent athletes. MethodsThe study group consisted of 28 adolescents aged 13 years (14 boys and 14 girls) from a sports secondary school. ResultsVEGF (vascular endothelial growth factor) serum concentration decreased after long-term training and changes were statistically significant. Furthermore, VEGF levels (before and after training) were found to be significantly lower in the group of male athletes compared to the group of female athletes. During the study period, similar changes were observed in bFGF (basic fibroblast growth factor) levels, but changes were less evident. The mean bFGF levels were distinctly higher in boys at every phase of the study.After an 8-month period of sports training, mean arterial pressure (MAP) and systolic (SBP) and diastolic (DBP) blood pressure decreased in the overall group. Statistically significant differences were observed in relation to DBP and MAP. ConclusionLong-term sports training in young athletes is associated with a decrease in circulating serum VEGF and bFGF at rest. Moreover, reductions in diastolic blood pressure and mean arterial pressure were also observed.

Abstract 378: Endothelial Cell Hypoxia-inducible Factor (HIF) Activity is Necessary for Right Ventricular Remodeling in a Murine Model of Chronic-hypoxic Pulmonary Hypertension

Introduction: Pulmonary hypertension induced myocardial remodeling is required to minimize RV wall stress as pulmonary arterial pressures increase. Myocardial angiogenesis is presumed to play an integral role in early RV remodeling, though molecular regulators remain undefined. Endothelial cell (EC) expression of HIF-1 is necessary for adaptive LV remodeling during chronic pressure overload, with loss of EC-specific HIF-1 activity associated with reduced LV capillary density. We hypothesized that EC HIF activity may also be necessary for adaptive RV remodeling and angiogenesis in a model of chronic RV pressure overload. Methods: We used a murine model of chronic hypoxic pulmonary hypertension (CH-PH) to induce RV remodeling. Conditional transgenic mice lacking expression of the obligate HIF-1α/HIF-2α co-factor ARNT (aryl hydrocarbon receptor nuclear translocator) in Tie2+ lineage cells ( ARNT fl/fl ; Tie2-Cre +/- ) and ARNT fl/fl ;Tie2-Cre -/- controls were exposed to normobaric hypoxia (10% FiO 2 ) for 1-5 weeks. We measured RV hypertrophy (RVH), RV systolic pressure (RVSP), and myocardial EC proliferation (flow cytometry) as a marker for sprouting angiogenesis. Results: After three weeks of CH-PH, RVH was observed in ARNT fl/fl ;Tie2-Cre -/- control mice, as anticipated (0.332±0.02 vs. 0.231±0.02; P &lt; 0.05). CH-PH induced RVH in ARNT fl/fl ;Tie2-Cre -/- mice was associated with an early (1 week) increase in RV EC proliferation (vs. LV, 4.5±1.3% vs.1.8±0.5%; P &lt; 0.05). Conversely, ARNT fl/fl ; Tie2 - Cre +/- mice did not develop RVH after 3 weeks of CH ( 0.222±0.01 vs. 0.241±0.02; P = NS), and early RV EC proliferation was attenuated (vs. LV, 2.9±1.2% vs. 1.5±0.3%; P = NS). Despite similar increases in CH-PH induced RVSP after 3 weeks, after 5 weeks RVSP normalized in ARNT fl/fl ; Tie2 - Cre +/- mice, but remained elevated in ARNT fl/fl ;Tie2-Cre -/- controls. Conclusions: These preliminary findings confirm that EC-specific HIF activity is necessary for CH-PH induced RVH, and suggest that reduced EC proliferation (and potentially angiogenesis) may contribute to abrogated RV remodeling in ARNT fl/fl ; Tie2 - Cre +/- mice. We hypothesize that late normalization of RVSP in these mice may be indicative of premature RV failure.

The effect of impaired angiogenesis on intestinal function following massive small bowel resection

PurposeIntestinal adaptation involves villus lengthening, crypt deepening, and increased capillary density following small bowel resection (SBR). Mice lacking the proangiogenic chemokine CXCL5 have normal structural adaptation but impaired angiogenesis. This work evaluates the impact of incomplete adaptive angiogenesis on the functional capacity of the intestine after SBR. MethodsCXCL5 knockout (KO) and C57BL/6 wild-type (WT) mice underwent 50% SBR. Magnetic resonance imaging measured weekly body composition. Intestinal absorptive capacity was evaluated through fecal fat analysis. Gene expression profiles for select macronutrient transporters were measured via RT-PCR. Postoperative crypt and villus measurements were assessed for structural adaptation. Submucosal capillary density was measured through CD31 immunohistochemistry. ResultsComparable postoperative weight gain occurred initially. Diminished weight gain, impaired fat absorption, and elevated steatorrhea occurred in KO mice after instituting high-fat diet. Greater postoperative upregulation of ABCA1 fat transporter occurred in WT mice, while PEPT1 protein transporter was significantly downregulated in KO mice. KO mice had impaired angiogenesis but intact structural adaptation. ConclusionAfter SBR, KO mice display an inefficient intestinal absorption profile with perturbed macronutrient transporter expression, impaired fat absorption, and slower postoperative weight gain. In addition to longer villi and deeper crypts, an intact angiogenic response may be required to achieve functional adaptation to SBR.

Expression of chemokine receptors on peripheral blood T cells in children with chronic kidney disease.

Chemokine receptors play a role in leukocyte recruitment, activation, and maintaining effector functions and regulate adaptive immune response and angiogenesis. The study aimed at flow cytometric analysis of T cell subsets with selected surface chemokine receptors (CCR4, CCR5, CCR7, CXCR3, and CXCR4) or receptor combination in peripheral blood of children with chronic kidney disease (CKD) on hemodialysis (HD). The percentage of T lymphocytes with CD8 and combined CD28,CCR7 expression was higher in HD children. The percentage of T lymphocytes expressing CCR7, CD28,CCR7, and CXCR4,CD8 was increased in children on conservative treatment. Total number (tn) of CXCR4+ cells was reduced in children on hemodialysis. The tn of T CXCR3+ cells was lower in children on conservative treatment. During HD the percentage of T CD4+ cells was higher and of T CXCR3+ lymphocytes was lower after HD session as compared to 15 min of session duration. During HD tn of T cells with expression of CCR4, CCR5, CCR7, CXCR3, and CXCR4 was constant. The alteration of chemokine receptors expression in children with CKD occurs early in the development. Diminished expression of CXCR3, CXCR4 on T cells in patients with CKD on HD might result in impaired inflammatory response. Increased CCR7+ T cell percentage could be responsible for the alteration of migration of cells into secondary lymphatic organs.

Coronary vessels and cardiac myocytes of middle-aged rats demonstrate regional sex-specific adaptation in response to postmyocardial infarction remodeling

BackgroundAn increasing body of evidence indicates that left ventricular (LV) remodeling, especially the degree of reactive myocardial hypertrophy after myocardial infarction (MI), differs in males and females. Surprisingly, to date, the sex-specific post-MI alterations of the coronary vasculature remain undetermined. Therefore, we tested the hypothesis that adaptive coronary arteriolar and capillary modifications occurring in response to reactive myocyte hypertrophy differ between middle-aged male and female post-MI rats.MethodsA large MI was induced in 12-month-old male (M-MI) and female (F-MI) Sprague–Dawley rats by ligation of the left coronary artery. Four weeks after surgery, rats with transmural infarctions, greater than 50% of the LV free wall (FW), were evaluated. Sham-operated male (M-Sham) and female (F-Sham) rats served as an age-matched controls.ResultsF-MI and M-MI rats had similar sized infarcts (61.3% ± 3.9% vs. 61.5% ± 1.2%) and scale of LV remodeling, as indicated analogous remodeling indices (1.41 ± 0.11 vs. 1.39 ± 0.09). The degree of reactive post-MI myocardial hypertrophy was adequate to normalize LV weight-to-body weight ratio in both sexes; however, the F-MI rats, in contrast to males, showed no myocyte enlargement in the LVFW epimyocardium. At the same time, a greater than 50% expansion of myocyte area in the male epimyocardium and in the female endomyocardium was accompanied by a 23% (P < 0.05) increase in capillary-to-myocyte ratio, indicative of adaptive angiogenesis. Based on arteriolar length density in post-MI hearts, the resistance vessels grew in the male LVFW as well as the septum by 24% and 29%, respectively. In contrast, in females, a significant (30%) expansion of arteriolar bed was limited only to the LVFW. Moreover, in F-MI rats, the enlargement of the arteriolar bed occurred predominantly in the vessels with diameters <30 μm, whereas in M-MI rats, a substantial (two- to threefold) increase in the density of larger arterioles (30 to 50 μm in diameter) was also documented.ConclusionOur data reveal that while both sexes have a relatively similar pattern of global LV remodeling and adaptive angiogenesis in response to a large MI, male and female middle-aged rats differ markedly in the regional scale of reactive cardiac myocyte hypertrophy and adaptive arteriogenesis.

Neuronal Control of Metabolism through Nutrient-Dependent Modulation of Tracheal Branching

SummaryDuring adaptive angiogenesis, a key process in the etiology and treatment of cancer and obesity, the vasculature changes to meet the metabolic needs of its target tissues. Although the cues governing vascular remodeling are not fully understood, target-derived signals are generally believed to underlie this process. Here, we identify an alternative mechanism by characterizing the previously unrecognized nutrient-dependent plasticity of the Drosophila tracheal system: a network of oxygen-delivering tubules developmentally akin to mammalian blood vessels. We find that this plasticity, particularly prominent in the intestine, drives—rather than responds to—metabolic change. Mechanistically, it is regulated by distinct populations of nutrient- and oxygen-responsive neurons that, through delivery of both local and systemic insulin- and VIP-like neuropeptides, sculpt the growth of specific tracheal subsets. Thus, we describe a novel mechanism by which nutritional cues modulate neuronal activity to give rise to organ-specific, long-lasting changes in vascular architecture.

Alpha-calcitonin gene-related peptide is protective against pressure overload-induced heart failure

The sensory neuropeptide, α-calcitonin gene-related peptide (α-CGRP) is protective against hypertension-induced heart damage and cardiac ischemia/reperfusion injury. To determine whether this neuropeptide is also cardioprotective in heart failure, this study examined whether the absence of α-CGRP exacerbated the adverse cardiac remodeling, dysfunction and mortality in pressure overload heart failure induced by transverse aortic constriction (TAC). Male α-CGRP knockout (KO) and wild type (WT) mice had TAC or sham surgery at day 0 and were studied on days 3, 14, 21, and 28. The survival rate of TAC α-CGRP KO mice was lower than the TAC WT mice over the duration of the protocol. Left ventricular α-CGRP content in TAC WT mice was higher at days 3, 14, and 21 than sham WT mice. Echocardiography demonstrated greater adverse cardiac remodeling and dysfunction in the TAC α-CGRP KO compared to the TAC WT mice. The lung/body weight ratios and left ventricular masses were higher in TAC α-CGRP KO compared to the TAC WT mice. While there was increased cardiac fibrosis in the TAC WT mice compared to shams, the TAC α-CGRP KO mice had markedly increased fibrosis above that of the TAC WT mice. TAC WT mice had greater cardiac inflammation, cell death, and adaptive angiogenesis compared to sham mice. Importantly, the TAC α-CGRP KO mice had greater inflammation, cell death, and attenuation of angiogenesis compared to TAC WT hearts. Thus, α-CGRP plays a significant protective role in TAC-induced heart failure which may be mediated by decreased inflammation, cell death, and fibrosis.

Cell-free carrier system for localized delivery of peripheral blood cell-derived engineered factor signaling: towards development of a one-step device for autologous angiogenic therapy

Spatiotemporally-controlled delivery of hypoxia-induced angiogenic factor mixtures has been identified by this group as a promising strategy for overcoming the limited ability of chronically ischemic tissues to generate adaptive angiogenesis. We previously developed an implantable, as well as an injectable system for delivering fibroblast-produced factors in vivo. Here, we identify peripheral blood cells (PBCs) as the ideal factor-providing candidates, due to their autologous nature, ease of harvest and ample supply, and investigate wound-simulating biochemical and biophysical environmental parameters that can be controlled to optimize PBC angiogenic activity. It was found that hypoxia (3% O2) significantly affected the expression of a range of angiogenesis-related factors including VEGF, angiogenin and thrombospondin-1, relative to the normoxic baseline. While all three factors underwent down-regulation over time under hypoxia, there was significant variation in the temporal profile of their expression. VEGF expression was also found to be dependent on cell-scaffold material composition, with fibrin stimulating production the most, followed by collagen and polystyrene. Cell-scaffold matrix stiffness was an additional important factor, as shown by higher VEGF protein levels when PBCs were cultured on stiff vs. compliant collagen hydrogel scaffolds. Engineered PBC-derived factor mixtures could be harvested within cell-free gel and microsphere carriers. The angiogenic effectiveness of factor-loaded carriers could be demonstrated by the ability of their releasates to induce endothelial cell tubule formation and directional migration in in vitro Matrigel assays, and microvessel sprouting in the aortic ring assay. To aid the clinical translation of this approach, we propose a device design that integrates this system, and enables one-step harvesting and delivering of angiogenic factor protein mixtures from autologous peripheral blood. This will facilitate the controlled release of these factors both at the bed-side, as an angiogenic therapy in wounds and peripheral ischemic tissue, as well as pre-, intra- and post-operatively as angiogenic support for central ischemic tissue, grafts, flaps and tissue engineered implants.

Injectable system for spatio-temporally controlled delivery of hypoxia-induced angiogenic signalling

While chronically ischaemic tissues are continuously exposed to hypoxia, the primary angiogenic stimulus, they fail to appropriately respond to it, as hypoxia-regulated angiogenic factor production gradually undergoes down-regulation, thus hindering adaptive angiogenesis. We have previously reported on two strategies for delivering on demand hypoxia-induced signalling (HIS) in vivo, namely, implanting living or non-viable hypoxic cell-matrix depots that actively produce factors or act as carriers of factors trapped within the matrix during in vitro pre-conditioning, respectively. This study aims to improve this approach through the development of a novel, injectable system for delivering cell-free matrix HIS-carriers. 3D spiral collagen constructs, comprising an inner cellular and outer acellular compartment, were cultured under hypoxia (5% O2). Cell-produced angiogenic factors (e.g. VEGF, FGF, PLGF, IL-8) were trapped within the nano-porous matrix of the acellular compartment as they radially diffused through it. The acellular matrix was mechanically fragmented into micro-fractions and added into a low temperature (5°C) thermo-responsive type I collagen solution, which underwent a collagen concentration-dependent solution-to-gel phase transition at 37°C. Levels of VEGF and IL-8, delivered from matrix fractions into media by diffusion through collagen sol–gel, were up-regulated by day 4 of hypoxic culture, peaked at day 8, and gradually declined towards the baseline by day 20, while FGF levels were stable over this period. Factors captured within matrix fractions were bioactive after 3months freeze storage, as shown by their ability to induce tubule formation in an in vitro angiogenesis assay. This system provides a minimally invasive, and repeatable, method for localised delivery of time-specific, cell-free HIS factor mixtures, as a tool for physiological induction of spatio-temporally controlled angiogenesis.

Evidence Implicating Peroxisome Proliferator-Activated Receptor-γ in the Pathogenesis of Preeclampsia

Preeclampsia, a major cause of maternal and perinatal mortality and morbidity, is thought to be attributed, in part, to impaired trophoblast invasion. Peroxisome proliferator-activated receptors are ligand-activated transcription factors expressed in trophoblasts, which regulate the expression of a number of genes involved in cell differentiation and proliferation. We investigated the effect of the administration of a peroxisome proliferator-activated receptor-γ antagonist during uncomplicated pregnancy in rats. Using an intraperitoneal miniosmotic pump, healthy pregnant rats were administered either vehicle or the peroxisome proliferator-activated receptor-γ-specific antagonist, T0070907 (1 mg/kg per day from gestational days 11-15). Rats treated with T0070907 developed key features of preeclampsia, including elevated mean arterial blood pressure, proteinuria, endothelial dysfunction, reduced pup weight, and increased platelet aggregation. T0070907-treated rats had reduced plasma vascular endothelial growth factor and increased plasma soluble fms-like tyrosine kinase 1. Furthermore, increases in total placental soluble fms-like tyrosine kinase 1 mRNA and fms-like tyrosine kinase 1 protein were also demonstrated, suggesting the placenta as the main contributor to the increased circulating levels of soluble fms-like tyrosine kinase 1. The labyrinthine trophoblast in the placentas of T0070907-treated rats were less differentiated, had increased cellular proliferation, and were strongly immunopositive for CD-31 staining, indicating adaptive angiogenesis. The present study suggests that peroxisome proliferator-activated receptor-γ may play a pivotal role in the progression of a healthy pregnancy and may critically regulate the risk of preeclampsia. These findings have important implications regarding the underlying etiology of preeclampsia and potential therapeutic targets.

Angiogenesis - may the force be with you!

Angiogenesis is defined as the formation of new blood vessels from pre-existing vessels, in particular capillaries, and is controlled by a complex balance between pro- and anti-angiogenic factors. It plays a crucial role during embryonic and fetal development, reparative processes such as wound healing, physiological challenges such as pregnancy, and following changes in metabolic demand, e.g. during exercise training of striated muscle. More than 70 diseases may have an angiogenic component to the pathology, with either excessive or insufficient capillary supply, although the vast majority of research to date has examined tumour angiogenesis and thus concentrated on angiogenesis inhibition. This $4 billion, largely industry-driven effort has sought chemical signals that may be blocked by pharmaceutical interventions. The ‘magic bullet’ approach can lead to quite remarkable effects in animal models, but has so far produced disappointing results in humans, where it is now recognised that a combination of therapeutic modalities is required. But what drives capillary growth in adaptive tissue remodelling? Physiological angiogenesis, exemplified by the response to muscle activity, appears more driven by mechanical signals. Following in the footsteps of Olga Hudlicka's pioneering work, we have started to elucidate the differential signalling involved in alternative ways of growing capillaries in skeletal muscle of rats and mice (Egginton, 2009). This involved developing approaches to isolate, as best as is possible with in vivo models, the mechanical signals relevant to muscle activity: increased shear stress (by vasodilators) and passive stretch of muscle fibres (by overload). Changes in endothelial structure and expression profiles for various growth factors, proteases, adhesion molecules and endogenous inhibitors revealed a differential role for NO production and matrix metalloproteinase (MMP) activity in splitting and sprouting forms of angiogenesis, respectively. These appear to be driven by mechanotransduction of haemodynamic forces by the endothelium, modulated by metabolic demands of the host tissue, with the angiogenesis promoter vascular endothelial growth factor (VEGF) playing a central role (Fig. 1). Figure 1 Chemotransduction of signals arising from increased muscle activity by the endothelium may lead to increased capillarisation by two distinct processes, sprouting and splitting forms of angiogenesis, as does mechanotransduction of altered haemodynamic ... The Copenhagen School was founded a hundred years ago by Johannes Lindhard and August Krogh to study various aspects of the body's response to exercise, which they reported in great detail and precise terms. Much of our understanding of human muscle physiology comes from the invasive studies conducted there over many years, repeatedly directing attention to hitherto unexplored avenues of enquiry, including one of the first papers showing that increased capillary supply was an adaptive response of muscle to endurance training in humans (Andersen & Henriksson, 1977). It is fitting, therefore, that the latest paper from that very productive heritage to be published in The Journal of Physiology continues this enlightened tradition (Hoier et al. 2010). Ylva Hellsten's group have adopted a different approach to isolating the signals involved in driving adaptive angiogenesis associated with muscle activity. They and others provide a good example of translation from animal (Gavin & Wagner, 2001) to human (Gustafsson et al. 2002) studies in showing the dynamic response to short bouts of intense exercise involved an angiogenic response. The key finding was that VEGF may be produced by myocytes. This prompted them to determine whether passive muscle training (i.e. movement with little or no EMG activity) may act as a stimulus for release of pro-angiogenic cytokines using a combination of muscle biopsies, interstitial fluid dialysis and cell culture. Training without working may sound like a couch-potato's dream, but this innovative approach demonstrated that both capillary density and the number of capillaries around a fibre were significantly enhanced after 2 weeks. Importantly, increased capillarisation occurred only in the trained leg, confirming a positive angiogenic effect of passive movement in these subjects. Interestingly, the contralateral leg is usually not refractory to change. In both animals and human studies of increased muscle activity, blood flow may increase in the contralateral limb due to shifting balance. However, this does not occur during passive movement, suggesting that blood-borne factors may be restricted to the ipsilateral limb, likely candidates being cytokines such as IL-6 or IL-8. The passive training model may therefore prove helpful for understanding which physiological factors are important for capillary growth in skeletal muscle. The novel observation that in habitually active, healthy individuals passive training led to alterations in angiogenic readouts and induced increased capillarisation is quite remarkable and clearly has therapeutic potential. It may be useful with inactive elderly individuals, peripheral arterial disease patients with exercise claudication, and for patients with enforced inactivity (e.g. knee injuries), to improve or maintain limb capillarisation.

Endothelial-Specific Transgenesis of TNFR2 Promotes Adaptive Arteriogenesis and Angiogenesis

We have previously shown that the tumor necrosis factor receptor 2 (TNFR2) protein is highly upregulated in vascular endothelium in response to ischemia, and a global deletion of TNFR2 in mice blunts ischemia-induced arteriogenesis and angiogenesis. However, the role of endothelial TNFR2 is not defined. In this study, we used endothelial cell (EC)-specific transgenesis of TNFR2 (TNFR2-TG) in mice to determine the in vivo function of TNFR2 in arteriogenesis and angiogenesis. In a femoral artery ligation model, TNFR2-TG mice had enhanced limb perfusion recovery and ischemic reserve capacity. TNFR2-TG mice also exhibited significantly enhanced arteriogenesis in the upper limb, whereas capillary formation and maturation in the lower limb were associated with reduction in cellular apoptosis and increased proliferation. Consistently, ischemia-induced TNFR2-dependent bone marrow tyrosine kinase in chromosome X-vascular endothelial growth factor receptor 2 proangiogenic signaling was augmented in TNFR2-TG mice. To further determine whether EC-expressed TNFR2 is sufficient to mediate ischemia-induced angiogenesis, we crossed TNFR2-TG with TNFR2-deficient mice to generate TNFR2-knockout (KO)/TG mice, in which only vascular ECs express TNFR2. The EC-expressed TNFR2 partially rescued the defects of TNFR2-KO in ischemia-induced angiogenic signaling and flow recovery. These in vivo data support a critical role for endothelial TNFR2 in ischemia-mediated adaptive angiogenesis. Therefore, specific expression and activation of TNFR2 in ECs may provide a novel strategy for the treatment of vascular diseases such as coronary artery disease and peripheral arterial disease.