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Peripheral arterial disease affects nearly 202 million individuals worldwide, sometimes leading to non-healing ulcers or limb amputations in severe cases. Genetically modified stem cells offer potential advantages for therapeutically inducing angiogenesis via augmented paracrine release mechanisms and tuned dynamic responses to environmental stimuli at disease sites. Here, we report the application of nanoparticle-induced CXCR4-overexpressing stem cells in a mouse hindlimb ischemia model. We found that CXCR4 overexpression improved stem cell survival, modulated inflammation in situ, and accelerated blood reperfusion. These effects, unexpectedly, led to complete limb salvage and skeletal muscle repair, markedly outperforming the efficacy of the conventional angiogenic factor control, VEGF. Importantly, assessment of CXCR4-overexpressing stem cells in vitro revealed that CXCR4 overexpression induced changes in paracrine signaling of stem cells, promoting a therapeutically desirable pro-angiogenic and anti-inflammatory phenotype. These results suggest that nanoparticle-induced CXCR4 overexpression may promote favorable phenotypic changes and therapeutic efficacy of stem cells in response to the ischemic environment.

The vascular endothelial cells (ECs) that line the inner layer of blood vessels are responsible for maintaining vascular homeostasis under physiological conditions. In the presence of disease or injury, ECs can become dysfunctional and contribute to a progressive decline in vascular health. ECs are constantly exposed to a variety of dynamic mechanical stimuli, including hemodynamic shear stress, pulsatile stretch, and passive signaling cues derived from the extracellular matrix. This review describes the molecular mechanisms by which ECs perceive and interpret these mechanical signals. The translational applications of mechanosensing are then discussed in the context of endothelial-to-mesenchymal transition and engineering of vascular grafts.

Although tissue engineering using human-induced pluripotent stem cells is a promising approach for treatment of cardiovascular diseases, some limiting factors include the survival, electrical integration, maturity, scalability, and immune response of three-dimensional (3D) engineered tissues. Here we discuss these important roadblocks facing the tissue engineering field and suggest potential approaches to overcome these challenges., In this Comment, Ngan Huang et al. discuss recent advances in cardiovascular tissue engineering and some of the main challenges that remain in translating these advances to the clinic. The authors propose future direction for the field to focus research efforts.

Background CEP (ω‐[2‐carboxyethyl]pyrrole) protein adducts are the end products of lipid oxidation associated with inflammation and have been implicated in the induction of angiogenesis in pathological conditions such as tissue ischemia. We synthesized small molecules derived from CEP protein adducts and evaluated the angiogenic effect of the CEP analog CEP 03 in the setting of peripheral arterial disease. Methods and Results The angiogenic effect of CEP 03 was assessed by in vitro analysis of primary human microvascular endothelial cell proliferation and tubelike formation in Matrigel (Corning). In the presence of CEP 03, proliferation of endothelial cells in vitro increased by 27±18% under hypoxic (1% O 2 ) conditions, reaching similar levels to that of VEGF A (vascular endothelial growth factor A) stimulation (22±10%), relative to the vehicle control treatment. A similar effect of CEP 03 was demonstrated in the increased number of tubelike branches in Matrigel, reaching >70% induction in hypoxia, compared with the vehicle control. The therapeutic potential of CEP 03 was further evaluated in a mouse model of peripheral arterial disease by quantification of blood perfusion recovery and capillary density. In the ischemic hind limb, treatment of CEP 03 encapsulated within Matrigel significantly enhanced blood perfusion by 2‐fold after 14 days compared with those treated with Matrigel alone. Moreover, these results concurred with histological finding that treatment of CEP 03 in Matrigel resulted in a significant increase in microvessel density compared with Matrigel alone. Conclusions Our data suggest that CEP 03 has a profound positive effect on angiogenesis and neovessel formation and thus has therapeutic potential for treatment of peripheral arterial disease.

Although skeletal muscle is highly regenerative following injury or disease, endogenous self-regeneration is severely impaired in conditions of volume traumatic muscle loss. Consequently, tissue engineering approach is a promising approach to regenerate skeletal muscle. Biological scaffolds serve as not only structural support for the promotion of cellular ingrowth, but they also impart potent modulatory signaling cues that may be beneficial for tissue regeneration. In this work, the progress of tissue engineering approaches for skeletal muscle engineering and regeneration is overviewed, with a focus on the techniques to create biomimetic engineered tissue using extracellular cues. These factors include mechanical and electrical stimulation, geometric patterning, and delivery of growth factors or other bioactive molecules. We further describe the progress of evaluating the therapeutic efficacy of these approaches in preclinical models of muscle injury.

Muscle regeneration can be permanently impaired by traumatic injuries, despite the high regenerative capacity of skeletal muscle. Implantation of engineered biomimetic scaffolds to the site of muscle ablation may serve as an attractive off-the-shelf therapeutic approach. The objective of the study was to histologically assess the therapeutic benefit of a three-dimensional spatially patterned collagen scaffold, in conjunction with rehabilitative exercise, for treatment of volumetric muscle loss. To mimic the physiologic organization of skeletal muscle, which is generally composed of myofibers aligned in parallel, three-dimensional parallel-aligned nanofibrillar collagen scaffolds were fabricated. When implanted into the ablated murine tibialis anterior muscle, the aligned nanofibrillar scaffolds, in conjunction with voluntary caged wheel exercise, significantly improved the density of perfused microvessels, in comparison to treatments of the randomly oriented nanofibrillar scaffold, decellularized scaffold, or in the untreated control group. The abundance of neuromuscular junctions was 19-fold higher when treated with aligned nanofibrillar scaffolds in conjunction with exercise, in comparison to treatment of aligned scaffold without exercise. Although, the density of de novo myofibers was not significantly improved by aligned scaffolds, regardless of exercise activity, the cross-sectional area of regenerating myofibers was increased by > 60% when treated with either aligned and randomly oriented scaffolds, in comparison to treatment of decellularized scaffold or untreated controls. These findings demonstrate that voluntary exercise improved the regenerative effect of aligned scaffolds by augmenting neurovascularization, and have important implications in the design of engineered biomimetic scaffolds for treatment of traumatic muscle injury.

Research Background and Objectives: Endothelial cells (ECs) are a promising cell type for the treatment of peripheral arterial disease (PAD). We previously showed that anisotropic nanofibrillar scaffolds augment the angiogenic function of ECs. Here we reported the effect of nanofibril size and crosslinking on EC survival and function to optimize the biophysical properties of scaffolds for treatment of PAD. Methods: Aligned nanofibrillar collagen scaffolds of varying fibril diameters and stiffness were prepared by altering the ionic strength (IS) of monomeric collagen and then crosslinked at varying levels of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Scaffolds were characterized for surface topography and nanofibril diameter by atomic force microscopy (AFM). Primary human ECs seeded onto the scaffolds were assessed for migration and survival under hypoxia. The regenerative potential of EC-seeded scaffolds was examined in a mouse hind limb ischemia (HLI) model. Results: Scaffold formulation with varying ionic strength (IS) resulted in different fibril diameter regardless of the degree of crosslinking ( Figure 1A: ~100 nm in low IS group vs. ~200 nm in high IS groups). When cultured under hypoxia, ECs seeded on high IS scaffold crosslinked by 1xEDC exhibited improved survival compared to those on low IS. Accordingly, the high IS 1xEDC scaffold was selectively tested in a mouse HLI model for cell survival and blood perfusion restoration. Bioluminescently-tagged ECs were seeded onto high IS 1xEDC scaffold or a randomly oriented fibrillar scaffold (20 mm of each). Non-invasive bioluminescence imaging for quantification of viable cells demonstrated markedly higher cell survival on aligned nanofibrillar scaffold than on randomly oriented scaffold ( Figure 1B, C ). Significance: Aligned nanofibrillar scaffolds that improve survival and angiogenesis of ECs provide a viable clinical strategy for the therapeutic neovascularization.

A key feature of peripheral arterial disease (PAD) is damage to endothelial cells (ECs), resulting in lower limb pain and restricted blood flow. Recent preclinical studies demonstrate that the transplantation of ECs via direct injection into the affected limb can result in significantly improved blood circulation. Unfortunately, the clinical application of this therapy has been limited by low cell viability and poor cell function. To address these limitations we have developed an injectable, recombinant hydrogel, termed SHIELD (Shear-thinning Hydrogel for Injectable Encapsulation and Long-term Delivery) for cell transplantation. SHIELD provides mechanical protection from cell membrane damage during syringe flow. Additionally, secondary in situ crosslinking provides a reinforcing network to improve cell retention, thereby augmenting the therapeutic benefit of cell therapy. In this study, we demonstrate the improved acute viability of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) following syringe injection delivery in SHIELD, compared to saline. Using a murine hind limb ischemia model of PAD, we demonstrate enhanced iPSC-EC retention in vivo and improved neovascularization of the ischemic limb based on arteriogenesis following transplantation of iPSC-ECs delivered in SHIELD.

Endothelial cells derived from human pluripotent stem cells are a promising cell type for enhancing angiogenesis in ischemic cardiovascular tissues. However, our understanding of microenvironmental factors that modulate the process of endothelial differentiation is limited. We examined the role of combinatorial extracellular matrix (ECM) proteins on endothelial differentiation systematically using an arrayed microscale platform. Human pluripotent stem cells were differentiated on the arrayed ECM microenvironments for 5 days. Combinatorial ECMs composed of collagen IV + heparan sulfate + laminin (CHL) or collagen IV + gelatin + heparan sulfate (CGH) demonstrated significantly higher expression of CD31, compared to single-factor ECMs. These results were corroborated by fluorescence activated cell sorting showing a 48% yield of CD31+/VE-cadherin+ cells on CHL, compared to 27% on matrigel. To elucidate the signaling mechanism, a gene expression time course revealed that VE-cadherin and FLK1 were upregulated in a dynamically similar manner as integrin subunit β3 (>50 fold). To demonstrate the functional importance of integrin β3 in promoting endothelial differentiation, the addition of neutralization antibody inhibited endothelial differentiation on CHL-modified dishes by >50%. These data suggest that optimal combinatorial ECMs enhance endothelial differentiation, compared to many single-factor ECMs, in part through an integrin β3-mediated pathway.

Biological approaches to augment angiogenesis are promising for treatment of peripheral arterial disease (PAD). We propose the use of scaffold-based modified mRNA (mmRNA) delivery as a favorable approach for transient, localized gene delivery. We hypothesized that hepatocyte growth factor (HGF) mmRNA-seeded nanofibrillar scaffolds will enable localized and temporally controlled delivery of mmRNA, leading to augmentation of angiogenesis in a murine model of PAD. To establish the efficacy of mmRNA therapy, mmRNA encoding green fluorescence protein (GFP) was used as a fluorescent reporter for quantification of transfection efficiency. Aligned nanofibrillar collagen scaffolds were loaded with mmRNA and lipofectamine transfection agent. The temporal kinetics of mmRNA release into media was measured by ribogreen assay. To determine the transfection efficiency, human fibroblasts were cultured on the aligned nanofibrillar scaffolds, or on tissue culture plastic, and the efficiency of transfection was measured for up to 7 days and assayed for GFP expression. Based on ribogreen assay, the cumulative release of GFP mmRNA over the course of 14 days was 235 ng/cm scaffold. In vitro transfection efficiency on aligned scaffolds (75%) was markedly higher than on tissue culture plastic (45%) after 24h. The persistence of cellular transfection as quantified by western blotting showed GFP expression >5 days post-transfection. Next, to demonstrate therapeutic efficacy for treatment of PAD, scaffolds releasing HGF or GFP mmRNA were transplanted to the site of the murine ischemic hindlimb. At the end of the 14 day experiment, laser Doppler spectroscopy showed that HGF mmRNA scaffold group had a higher mean perfusion ratio (0.32 ±0.10) than the GFP mmRNA scaffold group (0.23±0.14), suggesting that HGF-scaffolds improved blood perfusion. In summary, these data suggest that HGF mmRNA-releasing scaffolds marked improved blood perfusion in a murine model of PAD.

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

To translate recent advances in induced pluripotent stem cell biology to clinical regenerative medicine therapies, new strategies to control the co-delivery of cells and growth factors are needed. Building on our previous work designing Mixing-Induced Two-Component Hydrogels (MITCHs) from engineered proteins, here we develop protein–polyethylene glycol (PEG) hybrid hydrogels, MITCH-PEG, which form physical gels upon mixing for cell and growth factor co-delivery. MITCH-PEG is a mixture of C7, which is a linear, engineered protein containing seven repeats of the CC43 WW peptide domain (C), and 8-arm star-shaped PEG conjugated with either one or two repeats of a proline-rich peptide to each arm (P1 or P2, respectively). Both 20 kDa and 40 kDa star-shaped PEG variants were investigated, and all four PEG-peptide variants were able to undergo a sol–gel phase transition when mixed with the linear C7 protein at constant physiological conditions due to noncovalent hetero-dimerization between the C and P domains. Due to the dynamic nature of the C–P physical crosslinks, all four gels were observed to be reversibly shear-thinning and self-healing. The P2 variants exhibited higher storage moduli than the P1 variants, demonstrating the ability to tune the hydrogel bulk properties through a biomimetic peptide-avidity strategy. The 20 kDa PEG variants exhibited slower release of encapsulated vascular endothelial growth factor (VEGF), due to a decrease in hydrogel mesh size relative to the 40 kDa variants. Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) adopted a well-spread morphology within three-dimensional MITCH-PEG cultures, and MITCH-PEG provided significant protection from cell damage during ejection through a fine-gauge syringe needle. In a mouse hindlimb ischemia model of peripheral arterial disease, MITCH-PEG co-delivery of hiPSC-ECs and VEGF was found to reduce inflammation and promote muscle tissue regeneration compared to a saline control.

At present, little is known about how endothelial cells respond to spatial variations in fluid shear stress such as those that occur locally during embryonic development, at heart valve leaflets, and at sites of aneurysm formation. We built an impinging flow device that exposes endothelial cells to gradients of shear stress. Using this device, we investigated the response of microvascular endothelial cells to shear-stress gradients that ranged from 0 to a peak shear stress of 9–210 dyn/cm2. We observe that at high confluency, these cells migrate against the direction of fluid flow and concentrate in the region of maximum wall shear stress, whereas low-density microvascular endothelial cells that lack cell-cell contacts migrate in the flow direction. In addition, the cells align parallel to the flow at low wall shear stresses but orient perpendicularly to the flow direction above a critical threshold in local wall shear stress. Our observations suggest that endothelial cells are exquisitely sensitive to both magnitude and spatial gradients in wall shear stress. The impinging flow device provides a, to our knowledge, novel means to study endothelial cell migration and polarization in response to gradients in physical forces such as wall shear stress.

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

Background: Nitric oxide (NO), a potent vasodilator and anti-atherogenic molecule, is synthesized in various cell types, including vascular endothelial cells (ECs). The biological importance of NO enforces the need to develop and characterize specific and sensitive probes. To date, several fluorophores, chromophores and colorimetric techniques have been developed to detect NO or its metabolites (NO2 and NO3) in biological fluids, viable cells or cell lysates. Methods: Recently, a novel probe (NO550) has been developed and reported to detect NO in solutions and in primary astrocytes and neuronal cells with a fluorescence signal arising from a nonfluorescent background. Results: Here, we report further characterization of this probe by optimizing conditions for the detection and imaging of NO products in primary vascular ECs, fibroblasts, and embryonic stem cell- and induced pluripotent stem cell-derived ECs in the absence and presence of pharmacological agents that modulate NO levels. In addition, we studied the stability of this probe in cells over time and evaluated its compartmentalization in reference to organelle-labeling dyes. Finally, we synthesized an inherently fluorescent diazo ring compound (AZO550) that is expected to form when the nonfluorescent NO550 reacts with cellular NO, and compared its cellular distribution with that of NO550. Conclusion: NO550 is a promising agent for imaging NO at baseline and in response to pharmacological agents that modulate its levels.

Endothelial cells (ECs) are aligned longitudinally under laminar flow, whereas they are polygonal and poorly aligned in regions of disturbed flow. The unaligned ECs in disturbed flow fields manifest altered function and reduced survival that promote lesion formation. We demonstrate that the alignment of the ECs may directly influence their biology, independent of fluid flow. We developed aligned nanofibrillar collagen scaffolds that mimic the structure of collagen bundles in blood vessels, and examined the effects of these materials on EC alignment, function, and in vivo survival. ECs cultured on 30-nm diameter aligned fibrils re-organized their F-actin along the nanofibril direction, and were 50% less adhesive for monocytes than the ECs grown on randomly oriented fibrils. After EC transplantation into both subcutaneous tissue and the ischemic hindlimb, EC viability was enhanced when ECs were cultured and implanted on aligned nanofibrillar scaffolds, in contrast to non-patterned scaffolds. ECs derived from human induced pluripotent stem cells and cultured on aligned scaffolds also persisted for over 28 days, as assessed by bioluminescence imaging, when implanted in ischemic tissue. By contrast, ECs implanted on scaffolds without nanopatterning generated no detectable bioluminescent signal by day 4 in either normal or ischemic tissues. We demonstrate that 30-nm aligned nanofibrillar collagen scaffolds guide cellular organization, modulate endothelial inflammatory response, and enhance cell survival after implantation in normal and ischemic tissues.

Microscale and nanoscale structures can spatially pattern endothelial cells (ECs) into parallel-aligned organization, mimicking their cellular alignment in blood vessels exposed to laminar shear stress. However, the effects of spatial patterning on the function and global transcriptome of ECs are incompletely characterized. We used both parallel-aligned micropatterned and nanopatterned biomaterials to evaluate the effects of spatial patterning on the phenotype of ECs, based on gene expression profiling, functional characterization of monocyte adhesion, and quantification of cellular morphology. We demonstrate that both micropatterned and aligned nanofibrillar biomaterials could effectively guide EC organization along the direction of the micropatterned channels or nanofibrils, respectively. The ability of ECs to sense spatial patterning cues were abrogated in the presence of cytoskeletal disruption agents. Moreover, both micropatterned and aligned nanofibrillar substrates promoted an athero-resistant EC phenotype by reducing endothelial adhesiveness for monocytes and platelets, as well as by downregulating the expression of adhesion proteins and chemokines. We further found that micropatterned ECs have a transcriptional signature that is unique from non-patterned ECs, as well as from ECs aligned by shear stress. These findings highlight the importance of spatial patterning cues in guiding EC organization and function, which may have clinical relevance in the development of vascular grafts that promote patency.

Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iPSC-ECs) are promising for promoting angiogenesis in patients with cardiovascular disease, but a critical bottleneck is the ability to generate iPSC-ECs at high yields. Since endothelial cells physiologically interact with a milieu of basement membrane extracellular matrix (ECM) proteins, we aimed to examine the role of combinatorial ECMs in modulating endothelial differentiation using engineered ECM microenvironments. ECMs consisting of (gelatin (G), fibronectin (F), laminin (L), heparan sulfate proteoglycan (H), collagen IV (C), matrigel (M)) and the multi-component combinations thereof were conjugated onto glass slides to generate ECM microarrays composed of 63 unique combinatorial ECMs. Human iPSCs were differentiated on the ECM microarray for 5 days, and differentiation was quantified based on the degree of CD31 staining. Heat map analysis of CD31expression was significantly higher on G+M+L, compared to other combinations across 2 iPSC lines (Figure). RT-PCR further confirmed the upregulation in endothelial markers CD31 (>2-fold) and VE-cadherin (> 4-fold) when differentiated on G+M+L, compared to other ECMs. To elucidate the underlying mechanism, upregulation of endothelial genes were associated with upregulation of integrins (α1, α4, α5, αV, β1, β3, and β4) during the differentiation process. This data suggests a role of ECM-mediated integrin expression in modulating endothelial differentiation. Together, this data suggested that combinatorial ECMs differentially promote endothelial differentiation, in part through integrin-mediated pathways.

Since the heart is effectively an anisotropic organ in which the cardiomyocytes (CM) are locally aligned in series, it is important to engineer cardiac tissues that promote CM alignment in order to closely mimic the architecture of the native tissue, as well as better mimic the cellular composition of the heart. The objective of this study was to define the role of anisotropic extracellular matrix cues on the organization and survival of human induced pluripotent stem cell-derived CMs (hiPSC-CMs) by co-culturing hiPSC-CMs and primary endothelial cells (ECs) on parallel-aligned microfibrillar scaffolds. The hiPSC-CMs were generated from hiPSCs using small molecule Wnt pathway agonists and antagonists. Subsequently, the hiPSC-CMs were sequentially seeded on day 15 after EC attachment. We cultured monocultures and cocultures on electrospun three-dimensional (3D) scaffolds of polycaprolactone (PCL) and polyethylene oxide (PEO) polymer blends with an average fiber diameter of 14 μm. Aligned scaffolds were fabricated by stretching the randomly oriented scaffolds by 300% of the original scaffold length. Randomly oriented fibrillar scaffolds had an average pore diameter of 17 μm when compared to the 36 μm pore diameter of aligned scaffolds. Our results indicate that alignment of co-cultured cells at a 5:1 hiPSC-CMs : EC ratio was promoted by anistropic 3D electrospun scaffolds when compared to similar random 3D electrospun scaffolds. Additionally, cocultured cells on aligned fibrillar scaffolds had a mean angle of orientation of 30.8°, relative to the direction of fibrils, which was similar to that of hiPSC-CM monocultures on aligned scaffolds (32.8°). In contrast, the degree of alignment of hiPSC-CMs on randomly oriented fibrillary scaffolds was 43.4°, which suggests a non-oriented population of cells. Aligned scaffolds also produced more synchronized cardiomyocyte contraction than random scaffold orientations, although both induced spontaneous contraction frequency of ~1Hz. This study highlights the importance of nanotopographical cues and intercellular interactions in mediating the morphology and contractility of hiPSC-CMs for treatment of cardiovascular diseases such as myocardial infarction.

Secondary lymphedema is a common disorder associated with acquired functional impairment of the lymphatic system. The goal of this study was to evaluate the therapeutic efficacy of aligned nanofibrillar collagen scaffolds (BioBridge) positioned across the area of lymphatic obstruction in guiding lymphatic regeneration. In a porcine model of acquired lymphedema, animals were treated with BioBridge scaffolds, alone or in conjunction with autologous lymph node transfer as a source of endogenous lymphatic growth factor. They were compared with a surgical control group and a second control group in which the implanted BioBridge was supplemented with exogenous vascular endothelial growth factor-C (VEGF-C). Three months after implantation, immunofluorescence staining of lymphatic vessels demonstrated a significant increase in lymphatic collectors within close proximity to the scaffolds. To quantify the functional impact of scaffold implantation, bioimpedance was used as an early indicator of extracellular fluid accumulation. In comparison to the levels prior to implantation, the bioimpedance ratio was significantly improved only in the experimental BioBridge recipients with or without lymph node transfer, suggesting restoration of functional lymphatic drainage. These results further correlated with quantifiable lymphatic collectors, as visualized by contrast-enhanced computed tomography. They demonstrate the therapeutic potential of BioBridge scaffolds in secondary lymphedema.

The promise of cardiac tissue engineering is in the ability to recapitulate in vitro the functional aspects of a healthy heart and disease pathology as well as to design replacement muscle for clinical therapy. Parts of this promise have been realized; others have not. In a meeting of scientists in this field, five central challenges or “big questions” were articulated that, if addressed, could substantially advance the current state of the art in modeling heart disease and realizing heart repair.

Recent developments in cell therapy using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) hold great promise for treating ischemic cardiovascular tissues. However, poor post-transplantation viability largely limits the potential of stem cell therapy. Although the extracellular matrix (ECM) has become increasingly recognized as an important cell survival factor, conventional approaches primarily rely on single ECMs for in vivo co-delivery with cells, even though the endothelial basement membrane is comprised of a milieu of different ECMs. To address this limitation, we developed a combinatorial ECM microarray platform to simultaneously interrogate hundreds of micro-scale multi-component chemical compositions of ECMs on iPSC-EC response. After seeding iPSC-ECs onto ECM microarrays, we performed high-throughput analysis of the effects of combinatorial ECMs on iPSC-EC survival, endothelial phenotype, and nitric oxide production under conditions of hypoxia (1% O2) and reduced nutrients (1% fetal bovine serum), as is present in ischemic injury sites. Using automated image acquisition and analysis, we identified combinatorial ECMs such as collagen IV + gelatin + heparan sulfate + laminin and collagen IV + fibronectin + gelatin + heparan sulfate + laminin that significantly improved cell survival, nitric oxide production, and CD31 phenotypic expression, in comparison to single-component ECMs. These results were further validated in conventional cell culture platforms and within three-dimensional scaffolds. Furthermore, this approach revealed complex ECM interactions and non-intuitive cell behavior that otherwise could not be easily determined using conventional cell culture platforms. Together these data suggested that iPSC-EC delivery within optimal combinatorial ECMs may improve their survival and function under the condition of hypoxia with reduced nutrients. Statement of Significance Human endothelial cells (ECs) derived from induced pluripotent stem cells (iPSC-ECs) are promising for treating diseases associated with reduced nutrient and oxygen supply like heart failure. However, diminished iPSC-EC survival after implantation into diseased environments limits their therapeutic potential. Since native ECs interact with numerous extracellular matrix (ECM) proteins for functional maintenance, we hypothesized that combinatorial ECMs may improve cell survival and function under conditions of reduced oxygen and nutrients. We developed a high-throughput system for simultaneous screening of iPSC-ECs cultured on multi-component ECM combinations under the condition of hypoxia and reduced serum. Using automated image acquisition and analytical algorithms, we identified combinatorial ECMs that significantly improved cell survival and function, in comparison to single ECMs. Furthermore, this approach revealed complex ECM interactions and non-intuitive cell behavior that otherwise could not be easily determined.

The endothelium plays a pivotal role in vascular homeostasis, regulating the tone of the vascular wall, and its interaction with circulating blood elements. Alterations in endothelial functions facilitate the infiltration of inflammatory cells and permit vascular smooth muscle proliferation and platelet aggregation. Therefore, endothelial dysfunction is an early event in disease processes including atherosclerosis, and because of its critical role in vascular health the endothelium is worthy of the intense focus it has received. However, there are limitations to studying human endothelial function in vivo, or human vascular segments ex vivo. Thus, methods for endothelial cell culture have been developed and refined. More recently, methods to derive endothelial cells from pluripotent cells have extended the scientific range of human endothelial cell studies. Pluripotent stem cells may be generated, expanded and then differentiated into endothelial cells for in vitro studies. Constructs for molecular imaging can also be employed to facilitate tracking these cells in vivo. Furthermore, one can generate patient-specific endothelial cells to study the effects of genetic or epigenetic alterations on endothelial behavior. Finally, there is the opportunity to apply these cells for vascular therapy. This review focuses on the generation of endothelial cells from stem cells; their characterization by genetic, histological and functional studies; and their translational applications.

Stem cell-based therapeutics show promise for treatment of vascular diseases. However, the survival of the cells after in vivo injection into diseased tissues remains a concern. In the advent of non-invasive optical imaging techniques such as bioluminescence imaging (BLI), cell localization and survival can be easily monitored over time. This approach has recently been applied towards monitoring stem cell treatments for vascular regeneration of the coronary or peripheral arteries. In this review, we will describe the application of BLI for tracking transplanted stem cells and associating their viability with therapeutic efficacy, in preclinical disease models of vascular disease.

The role of nanotopographical extracellular matrix (ECM) cues in vascular endothelial cell (EC) organization and function is not well-understood, despite the composition of nano- to microscale fibrillar ECMs within blood vessels. Instead, the predominant modulator of EC organization and function is traditionally thought to be hemodynamic shear stress, in which uniform shear stress induces parallel-alignment of ECs with anti-inflammatory function, whereas disturbed flow induces a disorganized configuration with pro-inflammatory function. Since shear stress acts on ECs by applying a mechanical force concomitant with inducing spatial patterning of the cells, we sought to decouple the effects of shear stress using parallel-aligned nanofibrillar collagen films that induce parallel EC alignment prior to stimulation with disturbed flow resulting from spatial wall shear stress gradients. Using real time live-cell imaging, we tracked the alignment, migration trajectories, proliferation, and anti-inflammatory behavior of ECs when they were cultured on parallel-aligned or randomly oriented nanofibrillar films. Intriguingly, ECs cultured on aligned nanofibrillar films remained well-aligned and migrated predominantly along the direction of aligned nanofibrils, despite exposure to shear stress orthogonal to the direction of the aligned nanofibrils. Furthermore, in stark contrast to ECs cultured on randomly oriented films, ECs on aligned nanofibrillar films exposed to disturbed flow had significantly reduced inflammation and proliferation, while maintaining intact intercellular junctions. This work reveals fundamental insights into the importance of nanoscale ECM interactions in the maintenance of endothelial function. Importantly, it provides new insight into how ECs respond to opposing cues derived from nanotopography and mechanical shear force and has strong implications in the design of polymeric conduits and bioengineered tissues.

The extracellular matrix (ECM) microenvironment consists of structural and functional molecules. The ECM relays both biochemical and biophysical cues to and from the cells to modulate cell behavior and function. The biophysical cues can be engineered and applied to cells by means of spatial patterning, matrix rigidity, and matrix actuation. Tissue engineering strategies that utilize ECMs to direct stem cell organization and lineage specification show tremendous potential. This review describes the technologies for modulating ECM spatial patterning, matrix rigidity, chemical composition, and matrix actuation. The role of ECMs in vascular tissue engineering is then discussed as a model of tissue engineering and regenerative medicine.

Objective— We examined the effect of delivery modality on the survival, localization, and functional effects of exogenously administered embryonic stem cells (ESCs) or endothelial cells derived from them (ESC-ECs) in the ischemic hindlimb. Methods and Results— Murine ESCs or ESC-ECs were stably transduced with a construct for bioluminescence imaging (BLI) and fluorescent detection. In a syngeneic murine model of limb ischemia, ESCs or ESC-ECs were delivered by intramuscular (IM), intrafemoral artery (IA), or intrafemoral vein injections (n=5 in each group). For 2 weeks, cell survival and localization were tracked by BLI and confirmed by immunohistochemistry, and functional improvement was assessed by laser Doppler perfusion. BLI showed that ESCs localized to the ischemic limb after IM or IA, but not after intrafemoral vein administration. Regardless of the route of administration, ESCs were detected outside the hindlimb circulation in the spleen or lungs. ESCs did not improve limb perfusion and generated teratomas. In contrast, ESC-ECs delivered by all 3 modalities localized to the ischemic limb, as assessed by BLI. Most surprisingly, ESC-EC injected intrafemoral vein eventually localized to the ischemic limb after initially lodging in the pulmonary circulation. Immunohistochemical studies confirmed the engraftment of ESC-ECs into the limb vasculature after 2 weeks. Notably, ESC-ECs were not detected in the spleen or lungs after 2 weeks, regardless of route of administration. Furthermore, ESC-ECs significantly improved limb perfusion and neovascularization compared with the parental ESCs or the vehicle control group. Conclusion— In contrast to parental ESCs, ESC-ECs preferentially localized in the ischemic hindlimb by IA, IM, and intrafemoral vein delivery. ESC-ECs engrafted into the ischemic microvasculature, enhanced neovascularization, and improved limb perfusion.

Introduction: Stem cell injection is a minimally invasive approach for treatment of cardiovascular diseases including peripheral arterial disease (PAD), which affects over 8 million patients in the US. However, poor cell survival and low cell retention at injection site are critical bottlenecks to the efficacy of stem cell therapy. We developed a shear-thinning and self-healing hydrogel system with controllable rigidity for stem cell encapsulation so as to enhance transplant cell viability. Hypothesis: The injectable hydrogel system will prolong cell survival under ischemic conditions and maintain cellular phenotype, in comparison to cell injection in saline. Methods: We developed a hydrogel comprised of two complementary engineered proteins that self-assemble upon simple mixing. The hydrogel network incorporates a polyethylene glycol physical crosslinker that modulates hydrogel stiffness and degradation. Bioluminescently-labeled human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) were encapsulated within the hydrogel with controllable stiffness (G'~10-800 Pa) under conditions of hypoxia (1% O2). The cells within hydrogel were then subjected to an in vitro model of injection and assayed for cell survival, proliferation, and endothelial phenotype for up to 14 days. To verify these results in an experimental model of PAD, 10^6 cells were injected in saline or in 400 Pa hydrogel into the ischemic limb in SCID mice. Results: Acutely after injection, the survival of iPSC-ECs in saline was 65%, whereas survival of iPSC-ECs in hydrogels with stiffnesses of 10, 100, 400, and 800 Pa was 94%. Bioluminescence imaging of cell viability in hypoxia for 14 days demonstrated the highest proliferation in the hydrogel with 400 Pa stiffness. In the 400 Pa hydrogel, iPSC-ECs maintained elongated morphology with robust expression of endothelial phenotypic marker, CD31. In the ischemic hindlimb, iPSC-EC retention was markedly increased with hydrogel encapsulation, compared to saline delivery. Conclusions: These findings demonstrate that stem cell encapsulation within this protein hydrogel improves cell viability, which may have therapeutic benefit for treatment of PAD and broadly to myocardial ischemia.

Stem cell therapy is a promising approach for the treatment of tissue ischemia associated with myocardial infarction and peripheral arterial disease. Stem and progenitor cells derived from bone marrow or from pluripotent stem cells have shown therapeutic benefit in boosting angiogenesis as well as restoring tissue function. Notably, adult stem and progenitor cells including mononuclear cells, endothelial progenitor cells, and mesenchymal stem cells have progressed into clinical trials and have shown positive benefits. In this review, we overview the major classes of stem and progenitor cells, including pluripotent stem cells, and summarize the state of the art in applying these cell types for treating myocardial infarction and peripheral arterial disease.

Background The exact mechanism of stem cell therapy in augmenting the function of ischemic cardiomyopathy is unclear. In this study, we hypothesized that increased viability of the peri‐infarct region ( PIR ) produces restorative benefits after stem cell engraftment. A novel multimodality imaging approach simultaneously assessed myocardial viability (manganese‐enhanced magnetic resonance imaging [ MEMRI] ), myocardial scar (delayed gadolinium enhancement MRI ), and transplanted stem cell engraftment (positron emission tomography reporter gene) in the injured porcine hearts. Methods and Results Twelve adult swine underwent ischemia–reperfusion injury. Digital subtraction of MEMRI ‐negative myocardium (intrainfarct region) from delayed gadolinium enhancement MRI –positive myocardium ( PIR and intrainfarct region) clearly delineated the PIR in which the MEMRI ‐positive signal reflected PIR viability. Human amniotic mesenchymal stem cells ( hAMSC s) represent a unique population of immunomodulatory mesodermal stem cells that restored the murine PIR . Immediately following hAMSC delivery, MEMRI demonstrated an increased PIR viability signal compared with control. Direct PIR viability remained higher in hAMSC ‐treated hearts for >6 weeks. Increased PIR viability correlated with improved regional contractility, left ventricular ejection fraction, infarct size, and hAMSC engraftment, as confirmed by immunocytochemistry. Increased MEMRI and positron emission tomography reporter gene signal in the intrainfarct region and the PIR correlated with sustained functional augmentation (global and regional) within the hAMSC group (mean change, left ventricular ejection fraction: hAMSC 85±60%, control 8±10%; P hAMSC 24±8%, control 110±30%; P Conclusions The positron emission tomography reporter gene signal of hAMSC engraftment correlates with the improved MEMRI signal in the PIR . The increased MEMRI signal represents PIR viability and the restorative potential of the injured heart. This in vivo multimodality imaging platform represents a novel, real‐time method of tracking PIR viability and stem cell engraftment while providing a mechanistic explanation of the therapeutic efficacy of cardiovascular stem cells.

The objective of this study was to enhance the angiogenic capacity of endothelial cells (ECs) using nano-scale signaling cues from aligned nanofibrillar scaffolds in the setting of tissue ischemia. Thread-like nanofibrillar scaffolds with porous structure were fabricated from aligned-braided membranes generated under shear from liquid crystal collagen solution. Human ECs showed greater outgrowth from aligned scaffolds than from non-patterned scaffolds. Integrin α1 was in part responsible for the enhanced cellular outgrowth on aligned nanofibrillar scaffolds, as the effect was abrogated by integrin α1 inhibition. To test the efficacy of EC-seeded aligned nanofibrillar scaffolds in improving neovascularization in vivo, the ischemic limbs of mice were treated with: EC-seeded aligned nanofibrillar scaffold; EC-seeded non-patterned scaffold; ECs in saline; aligned nanofibrillar scaffold alone; or no treatment. After 14 days, laser Doppler blood spectroscopy demonstrated significant improvement in blood perfusion recovery when treated with EC-seeded aligned nanofibrillar scaffolds, in comparison to ECs in saline or no treatment. In ischemic hindlimbs treated with scaffolds seeded with human ECs derived from induced pluripotent stem cells (iPSC-ECs), single-walled carbon nanotube (SWNT) fluorophores were systemically delivered to quantify microvascular density after 28 days. Near infrared-II (NIR-II, 1000–1700 nm) imaging of SWNT fluorophores demonstrated that iPSC-EC-seeded aligned scaffolds group showed significantly higher microvascular density than the saline or cells groups. These data suggest that treatment with EC-seeded aligned nanofibrillar scaffolds improved blood perfusion and arteriogenesis, when compared to treatment with cells alone or scaffold alone, and have important implications in the design of therapeutic cell delivery strategies

With 25 million Americans suffering from at least one clinical manifestation of atherosclerosis, there is a pressing need to better understand the biological basis for the development of atherosclerosis. Endothelial cells (EC) that form the inner lining of blood vessels are primarily responsible for resisting atherosclerotic lesion formation. Although hemodynamic shear stress has been thought to be the dominant modulator of EC function, spatial patterning cues from nanoscale extracellular matrices (ECMs) that underlie the endothelium may play an equally important role. Therefore, the goal of this work was to examine how modulation of EC alignment using ECM nanopatterning cues modulates the formation of atherosclerosis. We hypothesized that ECs are more responsive to aligned nanopatterning cues than to disturbed flow, and can resist functional changes associated with atherosclerosis in the presence of disturbed flow. ECs were cultured on fibrillar scaffolds with nanotopography to either induce longitudinal alignment of ECs to mimic the morphology of healthy ECs, or to cause ECs to orient randomly (control) to mimic pathological ECs. ECs were then exposure to disturbed flow for 24 hours. Time-lapse microscopy and F-actin staining were used to quantify migration velocities and cellular orientation. ECs on aligned nanofibrillar scaffolds were induced to align along the direction of the fibrils, even after exposure to disturbed flow, and demonstrated a 2-fold decrease in migrational velocities, compared to randomly oriented ECs. This data suggests that nanoscale cues from aligned nanofibrillar scaffolds direct cell morphology and motility. Functional effects of nanofibrillar organization to resist inflammation, revealed a 2-fold decrease in monocyte adhesion on aligned ECs compared to randomly-oriented ECs, suggesting that ECs on aligned nanofibrillar scaffolds were less prone to atherosclerosis. These results were validated by analysis of inflammatory and shear-mediated markers (NFKB, ICAM, KLF2). This work reveals fundamental insights into the role of nanoscale ECM cues on early events of atherosclerosis, and has important translational potential in the generation of aligned nanofibrillar vascular grafts that resist lesion formation.

Hematopoietic stem cell (HSC) therapy for myocardial repair is limited by the number of stem cells that migrate to, engraft in, and proliferate at sites of injured myocardium. To alleviate this limitation, we studied whether a strategy using a bispecific antibody (BiAb) could target human stem cells specifically to injured myocardium and preserve myocardial function. Using a xenogeneic rat model whereby ischemic injury was induced by transient ligation of the left anterior descending artery (LAD), we determined the ability of a bispecific antibody to target human CD34+ cells to specific antigens expressed in ischemic injured myocardium. A bispecific antibody comprising an anti-CD45 antibody recognizing the common leukocyte antigen found on HSCs and an antibody recognizing myosin light chain, an organ-specific injury antigen expressed by infarcted myocardium, was prepared by chemical conjugation. CD34+ cells armed and unarmed with this BiAb were injected intravenously in rats 2 days postmyocardial injury. Immunohistochemistry studies showed that the armed CD34+ cells specifically localized to the infarcted region of the heart, colocalized with troponin T-stained cells, and colocalization with vascular structures. Compared to unarmed CD34+ cells, the bispecific antibody improved delivery of the stem cells to injured myocardium, and such targeted delivery was correlated with improved myocardial function 5 weeks after infarction (p < .01). Bispecific antibody targeting offers a unique means to improve the delivery of stem cells to facilitate organ repair and a tool to study stem cell biology.

We have developed a robust rat model of myocardial infarction (MI). Here we describe the step-by-step protocol for creating an ischemia-reperfusion rat model of MI. We also describe how to deliver therapeutic injections of mesenchymal stem cells (MSCs) together with fibrin, to show an application of this model. In addition, to confirm the presence of fibrin and cells in the infarct, visualization of MSCs and fibrin by histological techniques are also described. The ischemia-reperfusion MI model can be modified and generalized for use with various injectable polymers, cell types, drugs, DNA and combinations thereof. The model can be created in 7 days or less, depending on the timing of therapeutic intervention.

The migration of endothelial cells (ECs) plays an important role in vascular remodeling and regeneration. EC migration can be regulated by different mechanisms such as chemotaxis, haptotaxis, and mechanotaxis. This review will focus on fluid shear stress-induced mechanotransduction during EC migration. EC migration and mechanotransduction can be modulated by cytoskeleton, cell surface receptors such as integrins and proteoglycans, the chemical and physical properties of extracellular matrix (ECM) and cell-cell adhesions. The shear stress applied on the luminal surface of ECs can be sensed by cell membrane and associated receptor and transmitted throughout the cell to cell-ECM adhesions and cell-cell adhesions. As a result, shear stress induces directional migration of ECs by promoting lamellipodial protrusion and the formation of focal adhesions (FAs) at the front in the flow direction and the disassembly of FAs at the rear. Persistent EC migration in the flow direction can be driven by polarized activation of signaling molecules and the positive feedback loops constituted by Rho GTPases, cytoskeleton, and FAs at the leading edge. Furthermore, shear stress-induced EC migration can overcome the haptotaxis of ECs. Given the hemodynamic environment of the vascular system, mechanotransduction during EC migration has a significant impact on vascular development, angiogenesis, and vascular wound healing.

Novel strategies by which to repair ischemic myocardium after myocardial infarction include the use of three-dimensional polymer scaffolds. A comparative study was carried out to assess the therapeutic potential of fibrin, collagen I, and Matrigel as injectable biopolymers for repair after myocardial infarction. Using a rat model of left coronary artery occlusion followed by reperfusion, local injection of the biopolymers into the infarct zone yielded significantly higher levels of capillary formation, when compared with the saline control group, at 5 weeks posttreatment. However, the degree of angiogenesis was not significantly different among the biopolymers. In addition, the collagen biopolymer significantly enhanced infiltration of myofibroblasts into the infarct area when compared with the control group. The results of this study highlight the potential clinical benefit of these biopolymers as injectable scaffolds or cell delivery vehicles to the infarct zone after infarction.

Human embryonic stem (hES) cells hold promise as an unlimited source of cells for transplantation therapies. However, control of their proliferation and differentiation into complex, viable 3D tissues is challenging. Here we examine the use of biodegradable polymer scaffolds for promoting hES cell growth and differentiation and formation of 3D structures. We show that complex structures with features of various committed embryonic tissues can be generated, in vitro , by using early differentiating hES cells and further inducing their differentiation in a supportive 3D environment such as poly(lactic- co -glycolic acid)/poly( l -lactic acid) polymer scaffolds. We found that hES cell differentiation and organization can be influenced by the scaffold and directed by growth factors such as retinoic acid, transforming growth factor β, activin-A, or insulin-like growth factor. These growth factors induced differentiation into 3D structures with characteristics of developing neural tissues, cartilage, or liver, respectively. In addition, formation of a 3D vessel-like network was observed. When transplanted into severe combined immunodeficient mice, the constructs continue to express specific human proteins in defined differentiated structures and appear to recruit and anastamose with the host vasculature. This approach provides a unique culture system for addressing questions in cell and developmental biology, and provides a potential mechanism for creating viable human tissue structures for therapeutic applications.

Vascular endothelial cells (ECs) are longitudinally oriented in regions of laminar flow, but randomly distributed in regions of disturbed flow. The unaligned ECs in disturbed flow fields manifest altered function and reduced survival that promote atherosclerotic lesion formation. We hypothesized that the alignment of the ECs may directly influence their biology, independent of fluid flow. We fabricated parallel-aligned nanofibrillar collagen scaffolds with that mimic the native structure of collagen extracellular matrix within blood vessels, and examined the effects of aligned collagen nanopatterning on EC alignment, function, and in vivo survival. The 30-nm diameter aligned nanofibrils reoriented F-actin assembly along the nanofibril direction. ECs cultured on aligned nanofibrils were also 50% less adhesive for monocytes than the ECs grown on randomly oriented fibrils. To test the efficacy of the aligned nanofibrillar scaffolds in improving neovascularization in vivo, we induced unilateral hindlimb ischemia in SCID mice by excising the superficial femoral artery. The mice received one of the following treatments at the site of the excised femoral artery: 1) aligned nanofibrillar scaffold seeded with human ECs; 2) non-patterned scaffold seeded with ECs; 3) EC delivery in saline; or 4) no treatment (n>4). After 14 days, laser Doppler blood spectroscopy demonstrated significant improvement in blood perfusion in the group treated with cell-seeded aligned nanofibrillar scaffolds, in comparison to the groups with no treatment or EC delivery in saline. Furthermore, based on non-invasive bioluminescence imaging, the transplanted ECs persisted for longer periods of time when cultured on aligned nanofibrillar scaffolds, in comparison to non-patterned scaffolds. Together, these studies demonstrate that 30-nm aligned nanofibrillar collagen scaffolds guide cellular organization, modulate endothelial inflammatory response, and enhance cell survival and angiogenesis after implantation in the ischemic hind limb. These results have important implications for therapeutic cell delivery approaches.

Pericytes are perivascular cells localized to capillaries that promote vessel maturation, and their absence can contribute to vessel loss. Whether impaired endothelial–pericyte interaction contributes to small vessel loss in pulmonary arterial hypertension (PAH) is unclear. Using 3G5-specific, immunoglobulin G–coated magnetic beads, we isolated pericytes from the lungs of healthy subjects and PAH patients, followed by lineage validation. PAH pericytes seeded with healthy pulmonary microvascular endothelial cells failed to associate with endothelial tubes, resulting in smaller vascular networks compared to those with healthy pericytes. After the demonstration of abnormal polarization toward endothelium via live-imaging and wound-healing studies, we screened PAH pericytes for abnormalities in the Wnt/planar cell polarity (PCP) pathway, which has been shown to regulate cell motility and polarity in the pulmonary vasculature. PAH pericytes had reduced expression of frizzled 7 (Fzd7) and cdc42, genes crucial for Wnt/PCP activation. With simultaneous knockdown of Fzd7 and cdc42 in healthy pericytes in vitro and in a murine model of angiogenesis, motility and polarization toward pulmonary microvascular endothelial cells were reduced, whereas with restoration of both genes in PAH pericytes, endothelial–pericyte association was improved, with larger vascular networks. These studies suggest that the motility and polarity of pericytes during pulmonary angiogenesis are regulated by Wnt/PCP activation, which can be targeted to prevent vessel loss in PAH.

Cell-based approaches to improve blood perfusion are promising for treatment of peripheral arterial disease (PAD). However, cell delivery alone is limited by poor cell survival and the lack of organization among newly formed vessels. We hypothesized that parallel-aligned nanofibrillar scaffolds may guide endothelial cell (EC) assembly and accelerate the formation of organized vascular networks that enhance blood perfusion, in comparison to cell delivery in saline. We fabricated nanofibrillar collagen scaffolds with parallel-oriented configuration or randomly oriented configuration. Primary human ECs aligned along the direction of the nanofibrils of the aligned nanofibrillar scaffold, as shown by scanning electron microscopy, where the arrow denotes the orientation of nanofibrils (Fig 1). The ECs also had organized cytoskeleton within 20 degrees from the direction of the aligned nanofibrils. To test the efficacy of the aligned nanofibrillar scaffolds in improving neovascularization in vivo, we induced unilateral hindlimb ischemia in SCID mice by excising the superficial femoral artery. The mice received one of the following treatments at the site of the excised femoral artery: 1) aligned nanofibrillar scaffold seeded with human ECs; 2) randomly oriented nanofibrillar scaffold seeded with ECs; 3) EC delivery in saline; or 4) no treatment (n>4). After 14 days, laser Doppler blood spectroscopy demonstrated significant improvement in blood perfusion in the group treated with cell-seeded aligned nanofibrillar scaffolds, in comparison to the groups with no treatment or EC delivery in saline. In summary, these results suggest that aligned nanofibrillar scaffolds regulate EC morphology as well as angiogenic potential. This work highlights the innovative use of biomaterial nanopatterning as a way to promote blood perfusion, and has implications in the treatment of patients with PAD.

Over 8 million Americans are suffering from Peripheral Arterial Disease (PAD), which is due to atherosclerotic occlusion of the peripheral arteries of the limbs. Recent studies suggest that human induced pluripotent stem cell (iPSC)-derived endothelial cells (iPSC-ECs) can serve as a promising source for treatment of PAD. However, the low differentiation efficiency of iPSC-ECs is a major limitation to their clinical use. The purpose of this study was to investigate the role of extracellular matrix (ECM) and cell-ECM interactions in endothelial differentiation of iPSCs utilizing high throughput ECM microarray technique. We developed a high-throughput ECM microarray that contains circular printed areas of ECMs on glass slides using a robotic DNA microarrayer. Multi-component mixtures of purified ECMs components (gelatin, fibronectin, laminin, heparin sulfate proteoglycans, collagen IV, and matrigel) were deposited onto glass slides at fixed distances. A total of 280 spotted matrix features were fabricated w...

It is generally agreed that engineered cardiovascular tissues require cellular interactions with the local milieu. Within the microenvironment, the extracellular matrix (ECM) is an important support structure that provides dynamic signaling cues in part through its chemical, physical, and mechanical properties. In response to ECM factors, cells activate biochemical and mechanotransduction pathways that modulate their survival, growth, migration, differentiation, and function. This review describes the role of ECM chemical composition, spatial patterning, and mechanical stimulation in the specification of cardiovascular lineages, with a focus on stem cell differentiation, direct transdifferentiation, and endothelial-to-mesenchymal transition. The translational application of ECMs will be discussed in the context of cardiovascular tissue engineering and regenerative medicine.

This study examined the homing capacity of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) and their response to chemotactic gradients of stromal derived factor-1α (SDF). We have previously shown that EC derived from murine pluripotent stem cells can home to the ischemic hindlimb of the mouse. In the current study, we were interested to understand if ECs derived from human induced pluripotent stem cells are capable of homing. The homing capacity of iPSC-ECs was assessed after systemic delivery into immunodeficient mice with unilateral hindlimb ischemia. Furthermore, the iPSC-ECs were evaluated for their expression of CXCR4 and their ability to respond to SDF chemotactic gradients in vitro. Upon systemic delivery, the iPSC-ECs transiently localized to the lungs but did not home to the ischemic limb over the course of 14 days. To understand the mechanism of the lack of homing, the expression levels of the homing receptor, CXCR4, was examined at the transcriptional and protein levels. Furthermore, their ability to migrate in response to chemokines was assessed using microfluidic and scratch assays. Unlike ECs derived from syngeneic mouse pluripotent stem cells, human iPSC-ECs do not home to the ischemic mouse hindlimb. This lack of functional homing may represent an impairment of interspecies cellular communication or a difference in the differentiation state of the human iPSC-ECs. These results may have important implications in therapeutic delivery of iPSC-ECs.

Acute and sustained hypoxic pulmonary vasoconstriction (HPV), as well as chronic pulmonary hypertension (PH), is modulated by nitric oxide (NO). NO synthesis can be decreased by asymmetric dimethylarginine (ADMA), which is degraded by dimethylarginine dimethylaminohydrolase-1 (DDAH1). We investigated the effects of DDAH1 overexpression (DDAH1(tg)) on HPV and chronic hypoxia-induced PH. HPV was measured during acute (10 min) and sustained (3 h) hypoxia in isolated mouse lungs. Chronic PH was induced by the exposure of mice to 4 weeks of hypoxia. ADMA and cyclic 3',5'-guanosine monophosphate (cGMP) were determined by ELISA, and NO generation was determined by chemiluminescence. DDAH1 overexpression exerted no effects on acute HPV. However, DDAH1(tg) mice showed decreased sustained HPV compared with wild-type (WT) mice. Concomitantly, ADMA was decreased, and concentrations of NO and cGMP were significantly increased in DDAH1(tg). The administration of either Nω-nitro-l-arginine or 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one potentiated sustained HPV and partly abolished the differences in sustained HPV between WT and DDAH1(tg) mice. The overexpression of DDAH1 exerted no effect on the development of chronic hypoxia-induced PH. DDAH1 overexpression selectively decreased the sustained phase of HPV, partly via activation of the NO-cGMP pathway. Thus, increased ADMA concentrations modulate sustained HPV, but not acute HPV or chronic hypoxia-induced PH.

Objective— Transdifferentiation of fibroblasts to endothelial cells (ECs) may provide a novel therapeutic avenue for diseases, including ischemia and fibrosis. Here, we demonstrate that human fibroblasts can be transdifferentiated into functional ECs by using only 2 factors, Oct4 and Klf4, under inductive signaling conditions. Approach and Results— To determine whether human fibroblasts could be converted into ECs by transient expression of pluripotency factors, human neonatal fibroblasts were transduced with lentiviruses encoding Oct4 and Klf4 in the presence of soluble factors that promote the induction of an endothelial program. After 28 days, clusters of induced endothelial (iEnd) cells seemed and were isolated for further propagation and subsequent characterization. The iEnd cells resembled primary human ECs in their transcriptional signature by expressing endothelial phenotypic markers, such as CD31, vascular endothelial-cadherin, and von Willebrand Factor. Furthermore, the iEnd cells could incorporate acetylated low–density lipoprotein and form vascular structures in vitro and in vivo. When injected into the ischemic limb of mice, the iEnd cells engrafted, increased capillary density, and enhanced tissue perfusion. During the transdifferentiation process, the endogenous pluripotency network was not activated, suggesting that this process bypassed a pluripotent intermediate step. Conclusions— Pluripotent factor–induced transdifferentiation can be successfully applied for generating functional autologous ECs for therapeutic applications.

Manganese-Enhanced cardiac MRI (MEMRI) tracks long-term in vivo survival and restorative benefit of transplanted human Amnion-Derived Mesenchymal Stem Cells (hAMSC) after porcine ischemia-reperfusion injury Rajesh Dash, Paul J Kim, Yuka Matsuura, Xiaohu Ge, Fumiaki Ikeno, Jennifer K Lyons, Ngan F Huang, Scott Metzler, Patricia Nguyen, Shahriar Heidary, Marie-Claude Parent, Tomoaki Yamamoto, John Cooke, Pilar Ruiz-Lozano, Robert C Robbins, Joseph C Wu, Michael V McConnell, Alan Yeung, Phillip Harnish, Phillip C Yang

Tissue engineering and regenerative medicine hold tremendous promise for repairing diseased or dysfunctional tissues with functional biological replacements. To date, a number of engineered tissues are FDA-approved or are undergoing clinical trials. An important component of engineered tissues is the extracellular matrix (ECM) which provides mechanical and physical stability to the tissue, while also providing instructive signaling cues. In the advent of technological advancements, ECMs can be engineered with greater spatial and mechanical organization to better mimic physiological properties of native ECMs. The ECMs used in the generation of artificial skin and vascular grafts are highlighted as examples of engineered tissues under development and commercialization.