Physical activity energy expenditure and cardiometabolic health in three rural Kenyan populations.

OBJECTIVES: Physical activity is beneficial for metabolic health but the extent to which this may differ by ethnicity is still unclear. Here, the objective was to characterize the association between physical activity energy expenditure (PAEE) and cardiometabolic risk among the Luo, Kamba, and Maasai ethnic groups of rural Kenya. METHODS: In a cross-sectional study of 1084 rural Kenyans, free-living PAEE was objectively measured using individually-calibrated heart rate and movement sensing. A clustered metabolic syndrome risk score (zMS) was developed by averaging the sex-specific z-scores of five risk components measuring central adiposity, blood pressure, lipid levels, glucose tolerance, and insulin resistance. RESULTS: zMS was 0.08 (-0.09; -0.06) SD lower for every 10 kJ/kg/day difference in PAEE after adjustment for age and sex; this association was modified by ethnicity (interaction with PAEE P < 0.05). When adjusted for adiposity, each 10 kJ/kg/day difference in PAEE was predicted to lower zMS by 0.04 (-0.05, -0.03) SD, without evidence of interaction by ethnicity. The Maasai were predicted to have higher cardiometabolic risk than the Kamba and Luo at every quintile of PAEE, with a strong dose-dependent decreasing trend among all ethnicities. CONCLUSION: Free-living PAEE is strongly inversely associated with cardiometabolic risk in rural Kenyans. Differences between ethnic groups in this association were observed but were explained by differences in central adiposity. Therefore, targeted interventions to increase PAEE are more likely to be effective in subgroups with high central adiposity, such as Maasai with low levels of PAEE.

Transcriptional activation of hypoxia-inducible factor-1 (HIF-1) in myeloid cells promotes angiogenesis through VEGF and S100A8.

Emerging evidence indicates that myeloid cells are essential for promoting new blood vessel formation by secreting various angiogenic factors. Given that hypoxia-inducible factor (HIF) is a critical regulator for angiogenesis, we questioned whether HIF in myeloid cells also plays a role in promoting angiogenesis. To address this question, we generated a unique strain of myeloid-specific knockout mice targeting HIF pathways using human S100A8 as a myeloid-specific promoter. We observed that mutant mice where HIF-1 is transcriptionally activated in myeloid cells (by deletion of the von Hippel-Lindau gene) resulted in erythema, enhanced neovascularization in matrigel plugs, and increased production of vascular endothelial growth factor (VEGF) in the bone marrow, all of which were completely abrogated by either genetic or pharmacological inactivation of HIF-1. We further found that monocytes were the major effector producing VEGF and S100A8 proteins driving neovascularization in matrigel. Moreover, by using a mouse model of hindlimb ischemia we observed significantly improved blood flow in mice intramuscularly injected with HIF-1-activated monocytes. This study therefore demonstrates that HIF-1 activation in myeloid cells promotes angiogenesis through VEGF and S100A8 and that this may become an attractive therapeutic strategy to treat diseases with vascular defects.

Unexpected effect of proton pump inhibitors: elevation of the cardiovascular risk factor asymmetric dimethylarginine.

BACKGROUND: Proton pump inhibitors (PPIs) are gastric acid-suppressing agents widely prescribed for the treatment of gastroesophageal reflux disease. Recently, several studies in patients with acute coronary syndrome have raised the concern that use of PPIs in these patients may increase their risk of major adverse cardiovascular events. The mechanism of this possible adverse effect is not known. Whether the general population might also be at risk has not been addressed. METHODS AND RESULTS: Plasma asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase. Elevated plasma ADMA is associated with increased risk for cardiovascular disease, likely because of its attenuation of the vasoprotective effects of endothelial nitric oxide synthase. We find that PPIs elevate plasma ADMA levels and reduce nitric oxide levels and endothelium-dependent vasodilation in a murine model and ex vivo human tissues. PPIs increase ADMA because they bind to and inhibit dimethylarginine dimethylaminohydrolase, the enzyme that degrades ADMA. CONCLUSIONS: We present a plausible biological mechanism to explain the association of PPIs with increased major adverse cardiovascular events in patients with unstable coronary syndromes. Of concern, this adverse mechanism is also likely to extend to the general population using PPIs. This finding compels additional clinical investigations and pharmacovigilance directed toward understanding the cardiovascular risk associated with the use of the PPIs in the general population.

Conversion of human fibroblasts to functional endothelial cells by defined factors.

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.

Diagnosis and Management of Adult Malignancy-Associated Hemophagocytic Lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of severe, dysregulated inflammation driven by the inability of T cells to clear an antigenic target. When associated with malignancy (mHLH), the HLH syndrome is typically associated with extremely poor survival. Here, we review the diagnosis of secondary HLH (sHLH) syndromes in adults, with emphasis on the appropriate workup and treatment of mHLH. At present, the management of HLH in adults, including most forms of mHLH, is based on the use of corticosteroids and etoposide following the HLH-94 regimen. In some cases, this therapeutic approach may be cohesively incorporated into malignancy-directed therapy, while in other cases, the decision about whether to treat HLH prior to initiating other therapies may be more complicated. Recent studies exploring the efficacy of other agents in HLH, in particular ruxolitinib, offer hope for better outcomes in the management of mHLH. Considerations for the management of lymphoma-associated mHLH, as well as other forms of mHLH and immunotherapy treatment-related HLH, are discussed.

Endothelial cells derived from human iPSCS increase capillary density and improve perfusion in a mouse model of peripheral arterial disease.

OBJECTIVE: Stem cell therapy for angiogenesis and vascular regeneration has been investigated using adult or embryonic stem cells. In the present study, we investigated the potential of endothelial cells (ECs) derived from human induced pluripotent stem cells (hiPSCs) to promote the perfusion of ischemic tissue in a murine model of peripheral arterial disease. METHODS AND RESULTS: Endothelial differentiation was initiated by culturing hiPSCs for 14 days in differentiation media supplemented with BMP-4 and vascular endothelial growth factor. The hiPSC-ECs exhibited endothelial characteristics by forming capillary-like structures in matrigel and incorporating acetylated-LDL. They stained positively for EC markers such as KDR, CD31, CD144, and eNOS. In vitro exposure of hiPSC-ECs to hypoxia resulted in increased expression of various angiogenic related cytokines and growth factors. hiPSC-ECs were stably transduced with a double fusion construct encoded by the ubiquitin promoter, firefly luciferase for bioluminescence imaging and green fluorescence protein for fluorescent detection. The hiPSC-ECs (5×10(5)) were delivered by intramuscular injection into the ischemic hindlimb of SCID mice at day 0 and again on day 7 after femoral artery ligation (n=8). Bioluminescence imaging showed that hiPSC-ECs survived in the ischemic limb for at least 2 weeks. In addition, laser Doppler imaging showed that the ratio of blood perfusion was increased by hiPSC-EC treatment by comparison to the saline-treated group (0.58±0.12 versus 0.44±0.04; P=0.005). The total number of capillaries in the ischemic limb of mice receiving hiPSC-EC injections was greater than those in the saline-treated group (1284±155 versus 797±206 capillaries/mm(2)) (P<0.002). CONCLUSION: This study is a first step toward development of a regenerative strategy for peripheral arterial disease based on the use of ECs derived from hiPSCs.

Bacteroides fragilis-stimulated interleukin-10 contains expanding disease.

Commensal symbionts may become pathogens upon escaping their habitat. In the gut, Bacteroides fragilis protects against colitis through induction of interleukin 10 (IL-10) by CD4(+) T cells. When intestinal integrity is disrupted, B. fragilis and colonic contents escape into the peritoneum, causing abscesses and bacteremia. Whether the virulence mechanisms employed by B. fragilis during infections differ from those employed for symbiosis during commensalism is unknown. We demonstrate T cell-independent IL-10 production in response to B. fragilis during its pathogenic interactions with the host, and demonstrate the ability of the whole organism to activate Toll-like receptor 2-mediated MyD88 signaling in macrophages. Upon challenge with B. fragilis, mortality rates and serum proinflammatory cytokine levels were higher among IL-10(-/-) mice than among wild-type mice. Deaths were due to exuberant proinflammatory responses, not increased bacterial burden. During infection or commensalism, induction of IL-10 by B. fragilis is critical to this microbe's interactions with the immune system.

Agonizing over the Stimulatory Immune Checkpoint ICOS.

SUMMARY: Vopratelimab, an anti-ICOS (inducible costimulator of T cells) agonist, alone and in combination with nivolumab, possesses limited toxicity and modest clinical activity in a large phase I/II trial. This treatment induced ICOS expression of CD4+ T cells, which may enable biomarkers for patient selection. Nevertheless, T-cell agonists as cancer immunotherapies continue to be challenging. See related article by Yap et al., p. 3695.

Modulation of the local neutrophil response by a novel hyaluronic acid-binding peptide reduces bacterial burden during staphylococcal wound infection.

Novel approaches targeting the host's immune response to treat Staphylococcus aureus infections have significant potential to improve clinical outcomes, in particular during infection with antibiotic-resistant strains. The hyaluronic acid-binding peptide (HABP) PEP35 was assessed for its ability to treat S. aureus infections using a clinically relevant murine model of surgical wound infection. PEP35 demonstrated no direct antimicrobial activity against a range of antibiotic-susceptible and antibiotic-resistant clinical isolates of Staphylococcus aureus. However, when this peptide was administered at the onset of infection and up to 4 h postchallenge with a methicillin-susceptible (MSSA) or a methicillin-resistant (MRSA) strain of S. aureus, it significantly reduced the bacterial burden at the wound infection site. PEP35 reduced the tissue bacterial burden by exclusively modulating the local neutrophil response. PEP35 administration resulted in a significant early increase in local CXCL1 and CXCL2 production, which resulted in a more rapid influx of neutrophils to the infection site. Importantly, neutrophil influx was not sustained after treatment with PEP35, and administration of PEP35 alone did not induce a local inflammatory response. The immunomodulatory effects of PEP35 on CXC chemokine production were TLR2 and NF-κB dependent. We propose a novel role for a HABP as an innate immunomodulator in the treatment of MSSA and MRSA surgical wound infection through enhancement of the local CXC chemokine-driven neutrophil response.

Imaging of the epididymis.

Ultrasonography performed with a high-frequency transducer is the modality of choice for evaluating acute and nonacute scrotal disease. Acute epididymitis and epididymo-orchitis are the most common conditions that present with acute scrotal pain. Differentiation of these from testicular torsion is important for determining the appropriate management. High-transducer sonography allows the visualization of the epididymis and its detailed anatomy. We present important sonographic features of epididymitis and epididymo-orchitis caused by infectious and noninfectious etiologies. Features of benign and malignant epididymal lesions, including epididymal cysts, spermatoceles and tubular ectasia, sperm granulomas, adenomatoid tumors, leiomyomas, papillary cyst adenomas, lymphoma, and metastases are also presented. In addition, epididymal trauma and torsion are discussed. The goal of the review is to provide the radiologist with a better understanding of the numerous pathological conditions that occur in the epididymis.

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease.

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options. Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery. After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Population Effect of Differences in Cholesterol Guidelines in Eastern Europe and the United States.

IMPORTANCE: The American College of Cardiology/American Heart Association (ACC/AHA) guidelines for the management of blood cholesterol and the current European Society of Cardiology/European Atherosclerosis Society (ESC/EAS) guidelines differ in how they identify adults in need of statin therapy; furthermore, it is unclear how this difference translates into numbers and characteristics of patients recommended for treatment. OBJECTIVE: To determine the effect of the ACC/AHA and ESC/EAS cholesterol guidelines when applied to a population-based sample. DESIGN, SETTING, AND PARTICIPANTS: We used nationally representative data for 3055 adults aged 40 to 65 years from the 2007-2012 National Health and Nutrition Examination Surveys (NHANES) for the United States and for 1060 adults aged 40 to 65 years from the 2011 Nadcisnienie Tetnicze w Polsce survey for Poland. Data analysis was conducted from May 1, 2014, to December 31, 2015. MAIN OUTCOMES AND MEASURES: The number and characteristics of adults recommended for statin therapy according to the ACC/AHA and ESC/EAS guidelines were evaluated, and characteristics were compared between adults with discordant recommendations. RESULTS: The 3136 US adults in NHANES (2007-2012) aged 40 to 65 years represented 100.1 million adults; after excluding the 81 patients with missing data, these population estimates translate to 97.9 million adults. Similarly, the 1060 Polish adults in NATPOL (2011) aged 40 to 65 years represent 13.5 million adults. Using weighted data, in the United States, 43.8% (95% CI, 40.9%-46.7%) of adults would be recommended for statin therapy according to ACC/AHA guidelines and 39.1% (95% CI, 36.4%-41.8%) according to ESC/EAS guidelines. In Poland, 49.9% (95% CI, 46.9%-52.9%) of adults would be recommended for statin therapy under ACC/AHA guidelines compared with 47.6% (95% CI, 44.6%-50.7%) under ESC/EAS guidelines. Among individuals without cardiovascular disease and not currently taking statins, 11.0% of US and 10.5% of Polish adults had discordant guideline recommendations. Compared with individuals recommended for statin therapy by the ESC/EAS guidelines but not the ACC/AHA guidelines, those recommended for statin therapy under the ACC/AHA guidelines only had less chronic kidney disease; however, these individuals were also more likely to smoke, have lower high-density lipoprotein cholesterol levels, and have higher predicted 10-year risk of cardiovascular disease. CONCLUSIONS AND RELEVANCE: Despite differences in the ACC/AHA and EAS/ESC guidelines, the numbers of adults aged 40 to 65 years recommended for cholesterol-lowering therapy under each guideline were similar when applied to nationwide representative samples from both the United States and Poland. Discordant recommendations were driven by differences in the risk equations used in the 2 guidelines and different recommendations for adults with chronic kidney disease.

Patient-physician web messaging. The impact on message volume and satisfaction.

BACKGROUND: Patients want electronic access to providers. Providers fear being overwhelmed by unreimbursed messages. OBJECTIVE: Measure the effects of patient-physician web messaging on primary care practices. DESIGN/SETTING: Retrospective analysis of 6 case and 9 control internal medicine (IM) and family practice (FP) physicians' message volume, and a survey of 5,971 patients' web messaging with 267 providers and staff in 16 community primary care clinics in the Sacramento, CA region. MEASUREMENTS AND MAIN RESULTS: Case telephone volume was 18.2% lower (P=.002) and fell 6.50 times faster than control. Case total telephone plus web message volume was 13.7% lower (P=.025) and fell 5.84 times faster than control. Surveys were responded to by 40.3% (1,743/4,320) of patients and 61.4% (164/267) of providers and staff. Patients were overwhelmingly satisfied and providers and staff were generally satisfied; both found the system easy to use. Patient satisfaction correlated strongly with provider response time (Gamma=0.557), and provider/staff satisfaction with computer skills (Gamma=0.626) (Goodman-Kruskal Gamma [Gamma] measure of ordinal association). CONCLUSIONS: Secure web messaging improves on e-mail with encryption, access controls, message templates, customized message and prescription routing, knowledge content, and reimbursement. Further study is needed to determine whether reducing telephone traffic through the use of web messaging decreases provider interruptions and increases clinical efficiency during the workday. Satisfaction with web messaging may increase patient retention.

The impact of patient-physician Web messaging on provider productivity.

Patients increasingly want electronic access to providers, but physicians have been slow to offer e-access, fearing they will be overwhelmed by unreimbursed patient messages. This article reports on efforts to measure how patient-provider Web messaging affects physician productivity. To measure productivity, researchers analyzed the work of a group of physicians using a secure Web messaging system, compared with a control group that did not use the system. Results showed that physicians using the electronic media to communicate had about a 10 percent increase in productivity. Secure Web messaging's benefits also outweigh those of e-mail in increasing productivity and allaying concerns of physicians.

Aligned-Braided Nanofibrillar Scaffold with Endothelial Cells Enhances Arteriogenesis.

The objective of this study was to enhance the angiogenic capacity of endothelial cells (ECs) using nanoscale 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 nonpatterned 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 nonpatterned 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.

Near-infrared II fluorescence for imaging hindlimb vessel regeneration with dynamic tissue perfusion measurement.

BACKGROUND: Real-time vascular imaging that provides both anatomic and hemodynamic information could greatly facilitate the diagnosis of vascular diseases and provide accurate assessment of therapeutic effects. Here, we have developed a novel fluorescence-based all-optical method, named near-infrared II (NIR-II) fluorescence imaging, to image murine hindlimb vasculature and blood flow in an experimental model of peripheral arterial disease, by exploiting fluorescence in the NIR-II region (1000-1400 nm) of photon wavelengths. METHODS AND RESULTS: Because of the reduced photon scattering of NIR-II fluorescence compared with traditional NIR fluorescence imaging and thus much deeper penetration depth into the body, we demonstrated that the mouse hindlimb vasculature could be imaged with higher spatial resolution than in vivo microscopic computed tomography. Furthermore, imaging during 26 days revealed a significant increase in hindlimb microvascular density in response to experimentally induced ischemia within the first 8 days of the surgery (P<0.005), which was confirmed by histological analysis of microvascular density. Moreover, the tissue perfusion in the ischemic hindlimb could be quantitatively measured by the dynamic NIR-II method, revealing the temporal kinetics of blood flow recovery that resembled microbead-based blood flowmetry and laser Doppler blood spectroscopy. CONCLUSIONS: The penetration depth of millimeters, high spatial resolution, and fast acquisition rate of NIR-II imaging make it a useful imaging tool for murine models of vascular disease.

Chemotaxis of human induced pluripotent stem cell-derived endothelial cells.

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.

Human induced pluripotent stem cell-derived endothelial cells exhibit functional heterogeneity.

Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) are promising for treatment of vascular diseases. However, hiPSC-ECs purified based on CD31 expression are comprised of arterial, venous, and lymphatic subtypes. It is unclear whether hiPSC-ECs are heterogeneous in nature, and whether there may be functional benefits of enriching for specific subtypes. Therefore, we sought to characterize the hiPSC-ECs and enrich for each subtype, and demonstrate whether such enrichment would have functional significance. The hiPSC-ECs were generated from differentiation of hiPSCs using vascular endothelial growth factor (VEGF)-A and bone morphogenetic protein-4. The hiPSC-ECs were purified based on positive expression of CD31. Subsequently, we sought to enrich for each subtype. Arterial hiPSC-ECs were induced using higher concentrations of VEGF-A and 8-bromoadenosine-3':5'-cyclic monophosphate in the media, whereas lower concentrations of VEGF-A favored venous subtype. VEGF-C and angiopoietin-1 promoted the expression of lymphatic phenotype. Upon FACS purification based on CD31+ expression, the hiPSC-EC population was observed to display typical endothelial surface markers and functions. However, the hiPSC-EC population was heterogeneous in that they displayed arterial, venous, and to a lesser degree, lymphatic lineage markers. Upon comparing vascular formation in matrigel plugs in vivo, we observed that arterial enriched hiPSC-ECs formed a more extensive capillary network in this model, by comparison to a heterogeneous population of hiPSC-ECs. This study demonstrates that FACS purification of CD31+ hiPSC-ECs produces a diverse population of ECs. Refining the differentiation methods can enrich for subtype-specific hiPSC-ECs with functional benefits of enhancing neovascularization.

The modulation of endothelial cell morphology, function, and survival using anisotropic nanofibrillar collagen scaffolds.

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.