Integrating particle tracking with computational fluid dynamics to assess haemodynamic perturbation by coronary artery stents.

AIMS: Coronary artery stents have profound effects on arterial function by altering fluid flow mass transport and wall shear stress. We developed a new integrated methodology to analyse the effects of stents on mass transport and shear stress to inform the design of haemodynamically-favourable stents. METHODS AND RESULTS: Stents were deployed in model vessels followed by tracking of fluorescent particles under flow. Parallel analyses involved high-resolution micro-computed tomography scanning followed by computational fluid dynamics simulations to assess wall shear stress distribution. Several stent designs were analysed to assess whether the workflow was robust for diverse strut geometries. Stents had striking effects on fluid flow streamlines, flow separation or funnelling, and the accumulation of particles at areas of complex geometry that were tightly coupled to stent shape. CFD analysis revealed that stents had a major influence on wall shear stress magnitude, direction and distribution and this was highly sensitive to geometry. CONCLUSIONS: Integration of particle tracking with CFD allows assessment of fluid flow and shear stress in stented arteries in unprecedented detail. Deleterious flow perturbations, such as accumulation of particles at struts and non-physiological shear stress, were highly sensitive to individual stent geometry. Novel designs for stents should be tested for mass transport and shear stress which are important effectors of vascular health and repair.

The Movember Prostate Cancer Landscape Analysis: an assessment of unmet research needs.

Prostate cancer is a heterogeneous cancer with widely varying levels of morbidity and mortality. Approaches to prostate cancer screening, diagnosis, surveillance, treatment and management differ around the world. To identify the highest priority research needs across the prostate cancer biomedical research domain, Movember conducted a landscape analysis with the aim of maximizing the effect of future research investment through global collaborative efforts and partnerships. A global Landscape Analysis Committee (LAC) was established to act as an independent group of experts across urology, medical oncology, radiation oncology, radiology, pathology, translational research, health economics and patient advocacy. Men with prostate cancer and thought leaders from a variety of disciplines provided a range of key insights through a range of interviews. Insights were prioritized against predetermined criteria to understand the areas of greatest unmet need. From these efforts, 17 research needs in prostate cancer were agreed on and prioritized, and 3 received the maximum prioritization score by the LAC: first, to establish more sensitive and specific tests to improve disease screening and diagnosis; second, to develop indicators to better stratify low-risk prostate cancer for determining which men should go on active surveillance; and third, to integrate companion diagnostics into randomized clinical trials to enable prediction of treatment response. On the basis of the findings from the landscape analysis, Movember will now have an increased focus on addressing the specific research needs that have been identified, with particular investment in research efforts that reduce disease progression and lead to improved therapies for advanced prostate cancer.

Author Correction: Shear stress induces endothelial-to-mesenchymal transition via the transcription factor Snail.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mechanical Activation of Hypoxia-Inducible Factor 1α Drives Endothelial Dysfunction at Atheroprone Sites.

OBJECTIVE: Atherosclerosis develops near branches and bends of arteries that are exposed to low shear stress (mechanical drag). These sites are characterized by excessive endothelial cell (EC) proliferation and inflammation that promote lesion initiation. The transcription factor HIF1α (hypoxia-inducible factor 1α) is canonically activated by hypoxia and has a role in plaque neovascularization. We studied the influence of shear stress on HIF1α activation and the contribution of this noncanonical pathway to lesion initiation. APPROACH AND RESULTS: Quantitative polymerase chain reaction and en face staining revealed that HIF1α was expressed preferentially at low shear stress regions of porcine and murine arteries. Low shear stress induced HIF1α in cultured EC in the presence of atmospheric oxygen. The mechanism involves the transcription factor nuclear factor-κB that induced HIF1α transcripts and induction of the deubiquitinating enzyme Cezanne that stabilized HIF1α protein. Gene silencing revealed that HIF1α enhanced proliferation and inflammatory activation in EC exposed to low shear stress via induction of glycolysis enzymes. We validated this observation by imposing low shear stress in murine carotid arteries (partial ligation) that upregulated the expression of HIF1α, glycolysis enzymes, and inflammatory genes and enhanced EC proliferation. EC-specific genetic deletion of HIF1α in hypercholesterolemic apolipoprotein E-defecient mice reduced inflammation and endothelial proliferation in partially ligated arteries, indicating that HIF1α drives inflammation and vascular dysfunction at low shear stress regions. CONCLUSIONS: Mechanical low shear stress activates HIF1α at atheroprone regions of arteries via nuclear factor-κB and Cezanne. HIF1α promotes atherosclerosis initiation at these sites by inducing excessive EC proliferation and inflammation via the induction of glycolysis enzymes.

Shear stress induces endothelial-to-mesenchymal transition via the transcription factor Snail.

Blood flow influences atherosclerosis by generating wall shear stress, which alters endothelial cell (EC) physiology. Low shear stress induces dedifferentiation of EC through a process termed endothelial-to-mesenchymal transition (EndMT). The mechanisms underlying shear stress-regulation of EndMT are uncertain. Here we investigated the role of the transcription factor Snail in low shear stress-induced EndMT. Studies of cultured EC exposed to flow revealed that low shear stress induced Snail expression. Using gene silencing it was demonstrated that Snail positively regulated the expression of EndMT markers (Slug, N-cadherin, α-SMA) in EC exposed to low shear stress. Gene silencing also revealed that Snail enhanced the permeability of endothelial monolayers to macromolecules by promoting EC proliferation and migration. En face staining of the murine aorta or carotid arteries modified with flow-altering cuffs demonstrated that Snail was expressed preferentially at low shear stress sites that are predisposed to atherosclerosis. Snail was also expressed in EC overlying atherosclerotic plaques in coronary arteries from patients with ischemic heart disease implying a role in human arterial disease. We conclude that Snail is an essential driver of EndMT under low shear stress conditions and may promote early atherogenesis by enhancing vascular permeability.

Zebrafish Model for Functional Screening of Flow-Responsive Genes.

OBJECTIVE: Atherosclerosis is initiated at branches and bends of arteries exposed to disturbed blood flow that generates low shear stress. This mechanical environment promotes lesions by inducing endothelial cell (EC) apoptosis and dysfunction via mechanisms that are incompletely understood. Although transcriptome-based studies have identified multiple shear-responsive genes, most of them have an unknown function. To address this, we investigated whether zebrafish embryos can be used for functional screening of mechanosensitive genes that regulate EC apoptosis in mammalian arteries. APPROACH AND RESULTS: First, we demonstrated that flow regulates EC apoptosis in developing zebrafish vasculature. Specifically, suppression of blood flow in zebrafish embryos (by targeting cardiac troponin) enhanced that rate of EC apoptosis (≈10%) compared with controls exposed to flow (≈1%). A panel of candidate regulators of apoptosis were identified by transcriptome profiling of ECs from high and low shear stress regions of the porcine aorta. Genes that displayed the greatest differential expression and possessed 1 to 2 zebrafish orthologues were screened for the regulation of apoptosis in zebrafish vasculature exposed to flow or no-flow conditions using a knockdown approach. A phenotypic change was observed in 4 genes; p53-related protein (PERP) and programmed cell death 2-like protein functioned as positive regulators of apoptosis, whereas angiopoietin-like 4 and cadherin 13 were negative regulators. The regulation of perp, cdh13, angptl4, and pdcd2l by shear stress and the effects of perp and cdh13 on EC apoptosis were confirmed by studies of cultured EC exposed to flow. CONCLUSIONS: We conclude that a zebrafish model of flow manipulation coupled to gene knockdown can be used for functional screening of mechanosensitive genes in vascular ECs, thus providing potential therapeutic targets to prevent or treat endothelial injury at atheroprone sites.

Endothelial repair in stented arteries is accelerated by inhibition of Rho-associated protein kinase.

AIMS: Stent deployment causes endothelial cells (EC) denudation, which promotes in-stent restenosis and thrombosis. Thus endothelial regrowth in stented arteries is an important therapeutic goal. Stent struts modify local hemodynamics, however the effects of flow perturbation on EC injury and repair are incompletely understood. By studying the effects of stent struts on flow and EC migration, we identified an intervention that promotes endothelial repair in stented arteries. METHODS AND RESULTS: In vitro and in vivo models were developed to monitor endothelialization under flow and the influence of stent struts. A 2D parallel-plate flow chamber with 100 µm ridges arranged perpendicular to the flow was used. Live cell imaging coupled to computational fluid dynamic simulations revealed that EC migrate in the direction of flow upstream from the ridges but subsequently accumulate downstream from ridges at sites of bidirectional flow. The mechanism of EC trapping by bidirectional flow involved reduced migratory polarity associated with altered actin dynamics. Inhibition of Rho-associated protein kinase (ROCK) enhanced endothelialization of ridged surfaces by promoting migratory polarity under bidirectional flow (P < 0.01). To more closely mimic the in vivo situation, we cultured EC on the inner surface of polydimethylsiloxane tubing containing Coroflex Blue stents (65 µm struts) and monitored migration. ROCK inhibition significantly enhanced EC accumulation downstream from struts under flow (P < 0.05). We investigated the effects of ROCK inhibition on re-endothelialization in vivo using a porcine model of EC denudation and stent placement. En face staining and confocal microscopy revealed that inhibition of ROCK using fasudil (30 mg/day via osmotic minipump) significantly increased re-endothelialization of stented carotid arteries (P < 0.05). CONCLUSIONS: Stent struts delay endothelial repair by generating localized bidirectional flow which traps migrating EC. ROCK inhibitors accelerate endothelial repair of stented arteries by enhancing EC polarity and migration through regions of bidirectional flow.

TWIST1 Integrates Endothelial Responses to Flow in Vascular Dysfunction and Atherosclerosis.

RATIONALE: Blood flow-induced shear stress controls endothelial cell (EC) physiology during atherosclerosis via transcriptional mechanisms that are incompletely understood. The mechanosensitive transcription factor TWIST is expressed during embryogenesis, but its role in EC responses to shear stress and focal atherosclerosis is unknown. OBJECTIVE: To investigate whether TWIST regulates endothelial responses to shear stress during vascular dysfunction and atherosclerosis and compare TWIST function in vascular development and disease. METHODS AND RESULTS: The expression and function of TWIST1 was studied in EC in both developing vasculature and during the initiation of atherosclerosis. In zebrafish, twist was expressed in early embryonic vasculature where it promoted angiogenesis by inducing EC proliferation and migration. In adult porcine and murine arteries, TWIST1 was expressed preferentially at low shear stress regions as evidenced by quantitative polymerase chain reaction and en face staining. Moreover, studies of experimental murine carotid arteries and cultured EC revealed that TWIST1 was induced by low shear stress via a GATA4-dependent transcriptional mechanism. Gene silencing in cultured EC and EC-specific genetic deletion in mice demonstrated that TWIST1 promoted atherosclerosis by inducing inflammation and enhancing EC proliferation associated with vascular leakiness. CONCLUSIONS: TWIST expression promotes developmental angiogenesis by inducing EC proliferation and migration. In addition to its role in development, TWIST is expressed preferentially at low shear stress regions of adult arteries where it promotes atherosclerosis by inducing EC proliferation and inflammation. Thus, pleiotropic functions of TWIST control vascular disease and development.

Experimental Approaches to Study Endothelial Responses to Shear Stress.

SIGNIFICANCE: Shear stress controls multiple physiological processes in endothelial cells (ECs). RECENT ADVANCES: The response of ECs to shear has been studied using a range of in vitro and in vivo models. CRITICAL ISSUES: This article describes some of the experimental techniques that can be used to study endothelial responses to shear stress. It includes an appraisal of large animal, rodent, and zebrafish models of vascular mechanoresponsiveness. It also describes several bioreactors to apply flow to cells and physical methods to separate mechanoresponses from mass transport mechanisms. FUTURE DIRECTIONS: We conclude that combining in vitro and in vivo approaches can provide a detailed mechanistic view of vascular responses to force and that high-throughput systems are required for unbiased assessment of the function of shear-induced molecules. Antioxid. Redox Signal. 25, 389-400.

Computational fluid dynamics modelling in cardiovascular medicine.

This paper reviews the methods, benefits and challenges associated with the adoption and translation of computational fluid dynamics (CFD) modelling within cardiovascular medicine. CFD, a specialist area of mathematics and a branch of fluid mechanics, is used routinely in a diverse range of safety-critical engineering systems, which increasingly is being applied to the cardiovascular system. By facilitating rapid, economical, low-risk prototyping, CFD modelling has already revolutionised research and development of devices such as stents, valve prostheses, and ventricular assist devices. Combined with cardiovascular imaging, CFD simulation enables detailed characterisation of complex physiological pressure and flow fields and the computation of metrics which cannot be directly measured, for example, wall shear stress. CFD models are now being translated into clinical tools for physicians to use across the spectrum of coronary, valvular, congenital, myocardial and peripheral vascular diseases. CFD modelling is apposite for minimally-invasive patient assessment. Patient-specific (incorporating data unique to the individual) and multi-scale (combining models of different length- and time-scales) modelling enables individualised risk prediction and virtual treatment planning. This represents a significant departure from traditional dependence upon registry-based, population-averaged data. Model integration is progressively moving towards 'digital patient' or 'virtual physiological human' representations. When combined with population-scale numerical models, these models have the potential to reduce the cost, time and risk associated with clinical trials. The adoption of CFD modelling signals a new era in cardiovascular medicine. While potentially highly beneficial, a number of academic and commercial groups are addressing the associated methodological, regulatory, education- and service-related challenges.

Mechanoresponsive networks controlling vascular inflammation.

Atherosclerosis is a chronic inflammatory disease of arteries that develops preferentially at branches and bends that are exposed to disturbed blood flow. Vascular function is modified by flow, in part, via the generation of mechanical forces that alter multiple physiological processes in endothelial cells. Shear stress has profound effects on vascular inflammation; high uniform shear stress prevents leukocyte recruitment to the vascular wall by reducing endothelial expression of adhesion molecules and other inflammatory proteins, whereas low oscillatory shear stress has the opposite effects. Here, we review the molecular mechanisms that underpin the effects of shear stress on endothelial inflammatory responses. They include shear stress regulation of inflammatory mitogen-activated protein kinase and nuclear factor-κB signaling. High shear suppresses these pathways through the induction of several negative regulators of inflammation, whereas low shear promotes inflammatory signaling. Furthermore, we summarize recent studies indicating that inflammatory signaling is highly sensitive to pulse wave frequencies, magnitude, and direction of flow. Finally, the importance of systems biology approaches (including omics studies and functional screening) to identify novel mechanosensitive pathways is discussed.

Disturbed flow promotes endothelial senescence via a p53-dependent pathway.

OBJECTIVE: Although atherosclerosis is associated with systemic risk factors such as age, high cholesterol, and obesity, plaque formation occurs predominately at branches and bends that are exposed to disturbed patterns of blood flow. The molecular mechanisms that link disturbed flow-generated mechanical forces with arterial injury are uncertain. To illuminate them, we investigated the effects of flow on endothelial cell (EC) senescence. APPROACH AND RESULTS: LDLR(-/-) (low-density lipoprotein receptor(-/-)) mice were exposed to a high-fat diet for 2 to 12 weeks (or to a normal chow diet as a control) before the assessment of cellular senescence in aortic ECs. En face staining revealed that senescence-associated ß-galactosidase activity and p53 expression were elevated in ECs at sites of disturbed flow in response to a high-fat diet. By contrast, ECs exposed to undisturbed flow did not express senescence-associated ß-galactosidase or p53. Studies of aortae from healthy pigs (aged 6 months) also revealed enhanced senescence-associated ß-galactosidase staining at sites of disturbed flow. These data suggest that senescent ECs accumulate at disturbed flow sites during atherogenesis. We used in vitro flow systems to examine whether a causal relationship exists between flow and EC senescence. Exposure of cultured ECs to flow (using either an orbital shaker or a syringe-pump flow bioreactor) revealed that disturbed flow promoted EC senescence compared with static conditions, whereas undisturbed flow reduced senescence. Gene silencing studies demonstrated that disturbed flow induced EC senescence via a p53-p21 signaling pathway. Disturbed flow-induced senescent ECs exhibited reduced migration compared with nonsenescent ECs in a scratch wound closure assay, and thus may be defective for arterial repair. However, pharmacological activation of sirtuin 1 (using resveratrol or SRT1720) protected ECs from disturbed flow-induced senescence. CONCLUSIONS: Disturbed flow promotes endothelial senescence via a p53-p21-dependent pathway which can be inhibited by activation of sirtuin 1. These observations support the principle that pharmacological activation of sirtuin 1 may promote cardiovascular health by suppressing EC senescence at atheroprone sites.

Ischemic preconditioning for cell-based therapy and tissue engineering.

Cell- and tissue-based therapies are innovative strategies to repair and regenerate injured hearts. Despite major advances achieved in optimizing these strategies in terms of cell source and delivery method, the clinical outcome of cell-based therapy remains unsatisfactory. The non-genetic approach of ischemic/hypoxic preconditioning to enhance cell- and tissue-based therapies has received much attention in recent years due to its non-invasive drug-free application. Here we discuss the current development of hypoxic/ischemic preconditioning to enhance stem cell-based cardiac repair and regeneration.

The effects of stenting on shear stress: relevance to endothelial injury and repair.

Stent deployment following balloon angioplasty is used routinely to treat coronary artery disease. These interventions cause damage and loss of endothelial cells (EC), and thus promote in-stent thrombosis and restenosis. Injured arteries are repaired (intrinsically) by locally derived EC and by circulating endothelial progenitor cells which migrate and proliferate to re-populate denuded regions. However, re-endothelialization is not always complete and often dysfunctional. Moreover, the molecular and biomechanical mechanisms that control EC repair and function in stented segments are poorly understood. Here, we propose that stents modify endothelial repair processes, in part, by altering fluid shear stress, a mechanical force that influences EC migration and proliferation. A more detailed understanding of the biomechanical processes that control endothelial healing would provide a platform for the development of novel therapeutic approaches to minimize damage and promote vascular repair in stented arteries.

Hypoxic conditioning enhances the angiogenic paracrine activity of human adipose-derived stem cells.

Human adipose-derived stem cells (ASCs) secrete cytokines and growth factors that can be harnessed in a paracrine fashion for promotion of angiogenesis, cell survival, and activation of endogenous stem cells. We recently showed that hypoxia is a powerful stimulus for an angiogenic activity from ASCs in vitro and here we investigate the biological significance of this paracrine activity in an in vivo angiogenesis model. A single in vitro exposure of ASCs to severe hypoxia (<0.1% O2) significantly increased both the transcriptional and translational level of the vascular endothelial growth factor-A (VEGF-A) and angiogenin (ANG). The angiogenicity of the ASC-conditioned medium (ASC(CM)) was assessed by implanting ASC(CM)-treated polyvinyl alcohol sponges subcutaneously for 2 weeks in mice. The morphometric analysis of anti-CD31-immunolabeled sponge sections demonstrated an increased angiogenesis with hypoxic ASC(CM) treatment compared to normoxic control ASC(CM) treatment (percentage vascular volume; 6.0%±0.5% in the hypoxic ASC(CM) vs. 4.1%±0.7% in the normoxic ASC(CM), P<0.05). Reduction of VEGF-A and ANG levels in the ASC(CM) with respective neutralizing antibodies before sponge implantation showed a significantly diminished angiogenic response (3.5%±0.5% in anti-VEGF-A treated, 3.2%±0.7% in anti-ANG treated, and 3.5%±0.6% in anti-VEGF-A/ANG treated). Further, both the normoxic and hypoxic ASC(CM) were able to sustain in vivo lymphangiogenesis in sponges. Collectively, the model demonstrated that the increased paracrine production of the VEGF-A and ANG in hypoxic-conditioned ASCs in vitro translated to an in vivo effect with a favorable biological significance. These results further illustrate the potential for utilization of an in vitro optimized ASC(CM) for in vivo angiogenesis-related applications as an effective cell-free technology.

Differentiation of human adipose-derived stem cells into fat involves reactive oxygen species and Forkhead box O1 mediated upregulation of antioxidant enzymes.

Both reactive oxygen species (ROS) and Forkhead box O (FOXO) family transcription factors are involved in the regulation of adipogenic differentiation of preadipocytes and stem cells. While FOXO has a pivotal role in maintaining cellular redox homeostasis, the interactions between ROS and FOXO during adipogenesis are not clear. Here we examined how ROS and FOXO regulate adipogenesis in human adipose-derived stem cells (hASC). The identity of isolated cells was confirmed by their surface marker expression pattern typical for human mesenchymal stem cells (positive for CD29, CD44, CD73, CD90, and CD105, negative for CD45 and CD31). Using a standard adipogenic cocktail consisting of insulin, dexamethasone, indomethacin, and 3-Isobutyl-1-methylanxthine (IDII), adipogenesis was induced in hASC, which was accompanied by ROS generation. Scavenging ROS production with N-acetyl-L-cysteine or EUK-8, a catalytic mimetic of superoxide dismutase (SOD) and catalase, inhibited IDII-induced adipogenesis. We then mimicked IDII-induced oxidative stress through a lentiviral overexpression of Nox4 and an exogenous application of hydrogen peroxide in hASC and both manipulations significantly enhanced adipogenesis without changing the adipogenic differentiation rate. These data suggest that ROS promoted lipid accumulation in hASC undergoing adipogenesis. Antioxidant enzymes, including SOD2, catalase, and glutathione peroxidase were upregulated by IDII during adipogenesis, and these effects were blunted by FOXO1 silencing, which also suppressed significantly IDII-induced adipogenesis. Our findings demonstrated a balance of ROS generation and endogenous antioxidants in cells undergoing adipogenesis. Approaches targeting ROS and/or FOXO1 in adipocytes may bring new strategies to prevent and treat obesity and metabolic syndrome.

Enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro.

Long-term culture of primary neonatal rat cardiomyocytes is limited by the loss of spontaneous contractile phenotype within weeks in culture. This may be due to loss of contractile cardiomyocytes from the culture or overgrowth of the non-cardiomyocyte population. Using the mitochondria specific fluorescent dye, tetramethylrhodamine methyl ester perchlorate (TMRM), we showed that neonatal rat cardiomyocytes enriched by fluorescence-activated cell sorting can be maintained as contractile cultures for long periods (24-wk culture vs. 2 wk for unsorted cardiomyocytes). Long-term culture of this purified cardiomyocyte (TMRM high) population retained the expression of cardiomyocyte markers, continued calcium cycling, and displayed cyclic electrical activity that could be regulated pharmacologically. These findings suggest that non-cardiomyocyte populations can negatively influence contractility of cardiomyocytes in culture and that by purifying cardiomyocytes, the cultures retain potential as an experimental model for longitudinal studies of cardiomyocyte biology in vitro.

Transplantation of engineered cardiac muscle flaps in syngeneic rats.

Cardiac tissue engineering offers the prospect of a novel treatment for acquired or congenital heart defects. Previously, our studies have shown a significant mass of vascularized cardiac tissue can be generated using a vascularized tissue engineering chamber approach in nude rats. In this present study, syngeneic rats were investigated as an animal model for cardiac tissue engineering using the arteriovenous loop (AVL) chamber in the presence of a functional immune system. Neonatal cardiomyocytes implanted into the AVL chamber survived and assembled into a contractile flap confirming the basic features we previously showed in growing a cardiac construct. There was no significant loss of the assembled cardiac muscle from immune response. The engineered cardiac muscle flaps (ECMFs) formed were transplanted to the neck vessels of the same animal using a microsurgical technique, and all transplanted tissues remained contractile. The cardiac muscle volume of the control and transplant groups was estimated with histomorphometry using desmin and α-sarcomeric actin immunostaining, and there were no significant differences between the two groups. Finally, utilizing a novel model of transplantation, the ECMFs were transplanted to the heart of a recipient syngeneic rat as a vascularized tissue. The cardiac muscle within the transplanted ECMF was shown to survive and remain contractile for the 4-week post-transplantation period, and importantly, the cardiomyocytes retained the elongated, striated appearance of a mature phenotype. This study demonstrated the proof of concept for transplanting tissue-engineered cardiac muscle as a vascularized cardiac construct.

Ischemic preconditioning promotes intrinsic vascularization and enhances survival of implanted cells in an in vivo tissue engineering model.

Ischemic preconditioning (IPC) is a potent and effective means of protecting cells against ischemic injury. The protection has been demonstrated to involve release of paracrine factors that promote cell survival and angiogenesis, factors important for successful tissue engineering. The aim of the present study was to determine whether IPC of a vascular bed in vivo is an effective strategy to prepare it for tissue engineering with implanted cells. To test this hypothesis, an in vivo vascularized tissue engineering approach was employed, whereby polyacrylic chambers were placed around the femoral vessels of adult Sprague-Dawley rats. IPC was induced by 3 cycles of 5 min femoral artery occlusion interspersed with 5-min periods of reperfusion. Rats subjected to IPC generated bigger tissue constructs at 7 and 28 days postimplantation of empty chambers (∼50% increase in weight and volume, p<0.05). Morphometric counting of Masson trichrome stained tissue sections revealed significantly greater tissue construct volumes in ischemic preconditioned vascular beds at 7 and 28 days, increasing both fibrin matrix and vascularized tissue. Furthermore, morphometry of lectin-labeled blood vessels indicated an increase in vascular volume in IPC tissue constructs (∼100% increase vs. control, p<0.05). To investigate the cytoprotective effect of IPC, we implanted DiI-labeled neonatal rat cardiomyocytes in the chambers for 3 days, and IPC significantly reduced apoptosis of implanted cells as determined by the TUNEL assay and cleaved caspase-3 immunostaining. Furthermore, IPC significantly increased the cardiac muscle volume and vascular volume at 28 days after implantation of cardiomyocytes. In conclusion, in vivo IPC promotes survival of implanted cardiomyocytes and is associated with enhanced angiogenesis. IPC may represent a new approach to optimize tissue engineering with implanted cells.

Comparative analysis of paracrine factor expression in human adult mesenchymal stem cells derived from bone marrow, adipose, and dermal tissue.

Human adult mesenchymal stem cells (MSCs) support the engineering of functional tissue constructs by secreting angiogenic and cytoprotective factors, which act in a paracrine fashion to influence cell survival and vascularization. MSCs have been isolated from many different tissue sources, but little is known about how paracrine factor secretion varies between different MSC populations. We evaluated paracrine factor expression patterns in MSCs isolated from adipose tissue (ASCs), bone marrow (BMSCs), and dermal tissues [dermal sheath cells (DSCs) and dermal papilla cells (DPCs)]. Specifically, mRNA expression analysis identified insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor-D (VEGF-D), and interleukin-8 (IL-8) to be expressed at higher levels in ASCs compared with other MSC populations whereas VEGF-A, angiogenin, basic fibroblast growth factor (bFGF), and nerve growth factor (NGF) were expressed at comparable levels among the MSC populations examined. Analysis of conditioned media (CM) protein confirmed the comparable level of angiogenin and VEGF-A secretion in all MSC populations and showed that DSCs and DPCs produced significantly higher concentrations of leptin. Functional assays examining in vitro angiogenic paracrine activity showed that incubation of endothelial cells in ASC(CM) resulted in increased tubulogenic efficiency compared with that observed in DPC(CM). Using neutralizing antibodies we concluded that VEGF-A and VEGF-D were 2 of the major growth factors secreted by ASCs that supported endothelial tubulogenesis. The variation in paracrine factors of different MSC populations contributes to different levels of angiogenic activity and ASCs maybe preferred over other MSC populations for augmenting therapeutic approaches dependent upon angiogenesis.