Arteriolar dysgenesis in ischemic-regenerating skeletal muscle revealed by automated micromorphometry, computational modeling, and perfusion analysis.

Rebuilding the local vasculature is central to restoring the health of muscles subjected to ischemic injury. Arteriogenesis yields remodeled collateral arteries that circumvent the obstruction, and angiogenesis produces capillaries to perfuse the regenerating myofibers. However, the vital intervening network of arterioles that feed the regenerated capillaries is poorly understood and is an investigative challenge. We used machine learning and automated micromorphometry to quantify the arteriolar landscape in distal hindlimb muscles in mice that have regenerated after femoral artery excision. Assessment of 1,546 arteriolar sections revealed a striking (>2-fold) increase in arteriolar density in regenerated muscle 14 and 28 days after ischemic injury. Lumen caliber was initially similar to that of control arterioles but after 4 wk lumen area was reduced by 46%. In addition, the critical smooth muscle layer was attenuated throughout the arteriolar network, across a 150- to 5-µm diameter range. To understand the consequences of the reshaped distal hindlimb arterioles, we undertook computational flow modeling, which revealed blunted flow augmentation. Moreover, impaired flow reserve was confirmed in vivo by laser-Doppler analyses of flow in response to directly applied sodium nitroprusside. Thus, in hindlimb muscles regenerating after ischemic injury, the arteriolar network is amplified, inwardly remodels, and is diffusely undermuscularized. These defects and the associated flow restraints could contribute to the deleterious course of peripheral artery disease and merit attention when considering therapeutic innovations.NEW & NOTEWORTHY We report a digital pipeline for interrogating the landscape of arterioles in mouse skeletal muscle, using machine learning and automated micromorphometry. This revealed that in muscle regenerating after ischemic injury, the arteriolar density is increased but lumen caliber and smooth muscle content are reduced. Computational modeling and experimental validation reveal this arteriolar network to be functionally compromised, with diminished microvascular flow reserve.

Systematic Interrogation of Angiogenesis in the Ischemic Mouse Hind Limb: Vulnerabilities and Quality Assurance.

OBJECTIVE: There has been little success in translating preclinical studies of mouse hind limb ischemia into benefit for patients with peripheral artery disease. Using systematic strategies, we sought to define the injury and angiogenesis landscapes in mice subjected to hind limb ischemia and ascertain whether published studies to date have used an analysis strategy concordant with these data. Approach and Results: Maps of ischemic injury were generated from 22 different hind limb muscles and 33 muscle territories in 12-week-old C57BL/6 mice, based on loss or centralization of myofiber nuclei. Angiogenesis was similarly mapped based on CD (cluster of differentiation) 31-positive capillary content. Only 10 of 33 muscle territories displayed consistent muscle injury, with the distal anterior hind limb muscles most reliably injured. Angiogenesis was patchy and exclusively associated with zones of regenerated muscle (central nuclei). Angiogenesis was not observed in normal appearing muscle, necrotic muscle, or injury border zones. Systematic review of mouse hind limb angiogenesis studies identified 5147 unique publications, of which 509 met eligibility criteria for analysis. Only 7% of these analyzed manuscripts evaluated angiogenesis in distal anterior hind limb muscles and only 15% consistently examined for angiogenesis in zones of muscle regeneration. CONCLUSIONS: In 12-week C57BL/6 mice, angiogenesis postfemoral artery excision proceeds exclusively in zones of muscle regeneration. Only a minority of studies to date have analyzed angiogenesis in regions of demonstrably regenerating muscle or in high-likelihood territories. Quality assurance standards, informed by the atlas and mapping data herein, could augment data reliability and potentially help translate mouse hind limb ischemia studies to patient care.

Four-Dimensional Microvascular Analysis Reveals That Regenerative Angiogenesis in Ischemic Muscle Produces a Flawed Microcirculation.

RATIONALE: Angiogenesis occurs after ischemic injury to skeletal muscle, and enhancing this response has been a therapeutic goal. However, to appropriately deliver oxygen, a precisely organized and exquisitely responsive microcirculation must form. Whether these network attributes exist in a regenerated microcirculation is unknown, and methodologies for answering this have been lacking. OBJECTIVE: To develop 4-dimensional methodologies for elucidating microarchitecture and function of the reconstructed microcirculation in skeletal muscle. METHODS AND RESULTS: We established a model of complete microcirculatory regeneration after ischemia-induced obliteration in the mouse extensor digitorum longus muscle. Dynamic imaging of red blood cells revealed the regeneration of an extensive network of flowing neo-microvessels, which after 14 days structurally resembled that of uninjured muscle. However, the skeletal muscle remained hypoxic. Red blood cell transit analysis revealed slow and stalled flow in the regenerated capillaries and extensive arteriolar-venular shunting. Furthermore, spatial heterogeneity in capillary red cell transit was highly constrained, and red blood cell oxygen saturation was low and inappropriately variable. These abnormalities persisted to 120 days after injury. To determine whether the regenerated microcirculation could regulate flow, the muscle was subjected to local hypoxia using an oxygen-permeable membrane. Hypoxia promptly increased red cell velocity and flux in control capillaries, but in neocapillaries, the response was blunted. Three-dimensional confocal imaging revealed that neoarterioles were aberrantly covered by smooth muscle cells, with increased interprocess spacing and haphazard actin microfilament bundles. CONCLUSIONS: Despite robust neovascularization, the microcirculation formed by regenerative angiogenesis in skeletal muscle is profoundly flawed in both structure and function, with no evidence for normalizing over time. This network-level dysfunction must be recognized and overcome to advance regenerative approaches for ischemic disease.

Nicotinamide Phosphoribosyltransferase in Smooth Muscle Cells Maintains Genome Integrity, Resists Aortic Medial Degeneration, and Is Suppressed in Human Thoracic Aortic Aneurysm Disease.

RATIONALE: The thoracic aortic wall can degenerate over time with catastrophic consequences. Vascular smooth muscle cells (SMCs) can resist and repair artery damage, but their capacities decline with age and stress. Recently, cellular production of nicotinamide adenine dinucleotide (NAD+) via nicotinamide phosphoribosyltransferase (Nampt) has emerged as a mediator of cell vitality. However, a role for Nampt in aortic SMCs in vivo is unknown. OBJECTIVES: To determine whether a Nampt-NAD+ control system exists within the aortic media and is required for aortic health. METHODS AND RESULTS: Ascending aortas from patients with dilated aortopathy were immunostained for NAMPT, revealing an inverse relationship between SMC NAMPT content and aortic diameter. To determine whether a Nampt-NAD+ control system in SMCs impacts aortic integrity, mice with Nampt-deficient SMCs were generated. SMC-Nampt knockout mice were viable but with mildly dilated aortas that had a 43% reduction in NAD+ in the media. Infusion of angiotensin II led to aortic medial hemorrhage and dissection. SMCs were not apoptotic but displayed senescence associated-ß-galactosidase activity and upregulated p16, indicating premature senescence. Furthermore, there was evidence for oxidized DNA lesions, double-strand DNA strand breaks, and pronounced susceptibility to single-strand breakage. This was linked to suppressed poly(ADP-ribose) polymerase-1 activity and was reversible on resupplying NAD+ with nicotinamide riboside. Remarkably, we discovered unrepaired DNA strand breaks in SMCs within the human ascending aorta, which were specifically enriched in SMCs with low NAMPT. NAMPT promoter analysis revealed CpG hypermethylation within the dilated human thoracic aorta and in SMCs cultured from these tissues, which inversely correlated with NAMPT expression. CONCLUSIONS: The aortic media depends on an intrinsic NAD+ fueling system to protect against DNA damage and premature SMC senescence, with relevance to human thoracic aortopathy.

Segmentation of digitized histological sections for quantification of the muscularized vasculature in the mouse hind limb.

Immunohistochemical tissue staining enhances microvasculature characteristics, including the smooth muscle in the medial layer of the vessel walls that is responsible for regulation of blood flow. The vasculature can be imaged in a comprehensive fashion using whole-slide scanning. However, since each such image potentially contains hundreds of small vessels, manual vessel delineation and quantification is not practically feasible. In this work, we present a fully automatic segmentation and vasculature quantification algorithm for whole-slide images. We evaluated its performance on tissue samples drawn from the hind limbs of wild-type mice, stained for smooth muscle using 3,3'-Diaminobenzidine (DAB) immunostain. The algorithm was designed to be robust to vessel fragmentation due to staining irregularity, and artefactual staining of nonvessel objects. Colour deconvolution was used to isolate the DAB stain for detection of vessel wall fragments. Complete vessels were reconstructed from the fragments by joining endpoints of topological skeletons. Automatic measures of vessel density, perimeter, wall area and local wall thickness were taken. The segmentation algorithm was validated against manual measures, resulting in a Dice similarity coefficient of 89%. The relationships observed between these measures were as expected from a biological standpoint, providing further reinforcement of the accuracy of this system. This system provides a fully automated and accurate means of measuring the arteriolar and venular morphology of vascular smooth muscle.

Fibroblast Growth Factor 9 Imparts Hierarchy and Vasoreactivity to the Microcirculation of Renal Tumors and Suppresses Metastases.

Tumor vessel normalization has been proposed as a therapeutic paradigm. However, normal microvessels are hierarchical and vasoreactive with single file transit of red blood cells through capillaries. Such a network has not been identified in malignant tumors. We tested whether the chaotic tumor microcirculation could be reconfigured by the mesenchyme-selective growth factor, FGF9. Delivery of FGF9 to renal tumors in mice yielded microvessels that were covered by pericytes, smooth muscle cells, and a collagen-fortified basement membrane. This was associated with reduced pulmonary metastases. Intravital microvascular imaging revealed a haphazard web of channels in control tumors but a network of arterioles, bona fide capillaries, and venules in FGF9-expressing tumors. Moreover, whereas vasoreactivity was absent in control tumors, arterioles in FGF9-expressing tumors could constrict and dilate in response to adrenergic and nitric oxide releasing agents, respectively. These changes were accompanied by reduced hypoxia in the tumor core and reduced expression of the angiogenic factor VEGF-A. FGF9 was found to selectively amplify a population of PDGFRß-positive stromal cells in the tumor and blocking PDGFRß prevented microvascular differentiation by FGF9 and also worsened metastases. We conclude that harnessing local mesenchymal stromal cells with FGF9 can differentiate the tumor microvasculature to an extent not observed previously.

Type I collagen cleavage is essential for effective fibrotic repair after myocardial infarction.

Efficient deposition of type I collagen is fundamental to healing after myocardial infarction. Whether there is also a role for cleavage of type I collagen in infarct healing is unknown. To test this, we undertook coronary artery occlusion in mice with a targeted mutation (Col1a1(r/r)) that yields collagenase-resistant type I collagen. Eleven days after infarction, Col1a1(r/r) mice had a lower mean arterial pressure and peak left ventricular systolic pressure, reduced ventricular systolic function, and worse diastolic function, compared with wild-type littermates. Infarcted Col1a1(r/r) mice also had greater 30-day mortality, larger left ventricular lumens, and thinner infarct walls. Interestingly, the collagen fibril content within infarcts of mutant mice was not increased. However, circular polarization microscopy revealed impaired collagen fibril organization and mechanical testing indicated a predisposition to scar microdisruption. Three-dimensional lattices of collagenase-resistant fibrils underwent cell-mediated contraction, but the fibrils did not organize into birefringent collagen bundles. In addition, time-lapse microscopy revealed that, although cells migrated smoothly on wild-type collagen fibrils, crawling and repositioning on collagenase-resistant collagen was impaired. We conclude that type I collagen cleavage is required for efficient healing of myocardial infarcts and is critical for both dynamic positioning of collagen-producing cells and hierarchical assembly of collagen fibrils. This seemingly paradoxical requirement for collagen cleavage in fibrotic repair should be considered when designing potential strategies to inhibit matrix degradation in cardiac disease.

Directed differentiation of skin-derived precursors into functional vascular smooth muscle cells.

OBJECTIVE: The goal of this study was to characterize the factors and conditions required for smooth muscle cell (SMC)-directed differentiation of Sox2(+) multipotent rat and human skin-derived precursors (SKPs) and to define whether they represent a source of fully functional vascular SMCs for applications in vivo. METHODS AND RESULTS: We found that rat SKPs can differentiate almost exclusively into SMCs by reducing serum concentrations to 0.5% to 2% and plating them at low density. Human SKPs derived from foreskin required the addition of transforming growth factor-ß1 or -ß3 to differentiate into SMCs, but they did so even in the absence of serum. SMC formation was confirmed by quantitative reverse transcription-polymerase chain reaction, immunocytochemistry, and fluorescence-activated cell sorting, with increased expression of smoothelin-B and little to no expression of telokin or smooth muscle γ-actin, together indicating that SKPs differentiated into vascular rather than visceral SMCs. Rat and human SKP-derived SMCs were able to contract in vitro and also wrap around and support new capillary and larger blood vessel formation in angiogenesis assays in vivo. CONCLUSIONS: SKPs are Sox2(+) progenitors that represent an attainable autologous source of stem cells that can be easily differentiated into functional vascular SMCs in defined serum-free conditions without reprogramming. SKPs represent a clinically viable cell source for potential therapeutic applications in neovascularization.

Low-flow intussusception and metastable VEGFR2 signaling launch angiogenesis in ischemic muscle.

Efforts to promote sprouting angiogenesis in skeletal muscles of individuals with peripheral artery disease have not been clinically successful. We discovered that, contrary to the prevailing view, angiogenesis following ischemic muscle injury in mice was not driven by endothelial sprouting. Instead, real-time imaging revealed the emergence of wide-caliber, primordial conduits with ultralow flow that rapidly transformed into a hierarchical neocirculation by transluminal bridging and intussusception. This process was accelerated by inhibiting vascular endothelial growth factor receptor-2 (VEGFR2). We probed this response by developing the first live-cell model of transluminal endothelial bridging using microfluidics. Endothelial cells subjected to ultralow shear stress could reposition inside the flowing lumen as pillars. Moreover, the low-flow lumen proved to be a privileged location for endothelial cells with reduced VEGFR2 signaling capacity, as VEGFR2 mechanosignals were boosted. These findings redefine regenerative angiogenesis in muscle as an intussusceptive process and uncover a basis for its launch.

Obstruction of Small Arterioles in Patients with Critical Limb Ischemia due to Partial Endothelial-to-Mesenchymal Transition.

Critical limb ischemia (CLI) is a hazardous manifestation of atherosclerosis and treatment failure is common. Abnormalities in the arterioles might underlie this failure but the cellular pathobiology of microvessels in CLI is poorly understood. We analyzed 349 intramuscular arterioles in lower limb specimens from individuals with and without CLI. Arteriolar densities were 1.8-fold higher in CLI muscles. However, 33% of small (<20 µm) arterioles were stenotic and 9% were completely occluded. The lumens were closed by bulky, re-oriented endothelial cells expressing abundant N-cadherin that uniquely localized between adjacent and opposing endothelial cells. S100A4 and SNAIL1 were also expressed, supporting an endothelial-to-mesenchymal transition. SMAD2/3 was activated in occlusive endothelial cells and TGFß1 was increased in the adjacent mural cells. These findings identify a microvascular closure process based on mesenchymal transitions in a hyper-TGFß environment that may, in part, explain the limited success of peripheral artery revascularization procedures.

3D vessel-wall virtual histology of whole-body perfused mice using a novel heavy element stain.

Virtual histology - utilizing high-resolution three-dimensional imaging - is becoming readily available. Micro-computed tomography (micro-CT) is widely available and is often coupled with x-ray attenuating histological stains that mark specific tissue components for 3D virtual histology. In this study we describe a new tri-element x-ray attenuating stain and perfusion protocol that provides micro-CT contrast of the entire vasculature of an intact mouse. The stain - derived from an established histology stain (Verhoeff's) - is modified to enable perfusion through the vasculature; the attenuating elements of the stain are iodine, aluminum, and iron. After a 30-minute perfusion through the vasculature (10-minute flushing with detergent-containing saline followed by 15-minute perfusion with the stain and a final 5-minute saline flush), animals are scanned using micro-CT. We demonstrate that the new staining protocol enables sharp delineation of the vessel walls in three dimensions over the whole body; corresponding histological analysis verified that the CT stain is localized primarily in the endothelial cells and media of large arteries and the endothelium of smaller vessels, such as the coronaries. The rapid perfusion and scanning protocol ensured that all tissues are available for further analysis via higher resolution CT of smaller sections or traditional histological sectioning.

Fortifying Angiogenesis in Ischemic Muscle with FGF9-Loaded Electrospun Poly(Ester Amide) Fibers.

Delivery of angiogenic growth factors lessens ischemia in preclinical models but has demonstrated little benefit in patients with peripheral vascular disease. Augmenting the wrapping of nascent microvessels by mural cells constitutes an alternative strategy to regenerating a functional microvasculature, particularly if integrated with a sustained delivery platform. Herein, electrospun poly(ester amide) (PEA) nanofiber mats are fabricated for delivering a mural cell-targeting factor, fibroblast growth factor 9 (FGF9). Proof-of-principle is established by placing FGF9/FGF2-loaded PEA fiber mats on the chick chorioallantoic membrane and identifying enhanced angiogenesis by 3D power Doppler micro-ultrasound imaging. To assess the delivery system in ischemic muscle, FGF9-loaded PEA fiber mats are implanted onto the surface of the tibialis anterior muscle of mice with hindlimb ischemia. The system supplies FGF9 into the tibialis anterior muscle and yields a neo-microvascular network with enhanced mural cell coverage up to 28 days after injury. The regenerating muscle that receives FGF9 display near-normal sized myofibers and reduced interstitial fibrosis. Moreover, the mice demonstrate improved locomotion. These findings of locally released FGF9 from PEA nanofibers raise prospects for a microvascular remodeling approach to improve muscle health in peripheral vascular disease.

Vitamin D intervention does not improve vascular regeneration in diet-induced obese male mice with peripheral ischemia.

Vitamin D appears to either promote or inhibit neovascularization in a disease context-dependent manner. The effects of vitamin D, alone or in combination with niacin, on endothelial cell (EC) angiogenic function and on revascularization in obese animals with peripheral ischemia are unknown. Here, we report that supplementation of high palmitate medium with vitamin D, niacin or both vitamins increased EC tube formation, which relies primarily on cell migration, and also maintained tube stability over time. Transcriptomic analyses revealed that both vitamins increased stress response and anti-inflammatory gene expression. However, vitamin D decreased cell cycle gene expression and inhibited proliferation, while niacin induced stable expression of miR-126-3p and -5p and maintained cell proliferation in high palmitate. To assess vascular regeneration, diet-induced obese mice received vitamin D, niacin or both vitamins following hind limb ischemic injury. Niacin, but not vitamin D or combined treatment, improved recovery of hind limb use. Histology of tibialis anterior sections revealed no improvements in revascularization, regeneration, inflammation or fibrosis with vitamin D or combined treatment. In summary, although both vitamin D and niacin increased angiogenic function of EC cultures in high fat, only niacin improved recovery of hind limb use following ischemic injury in obese mice. It is possible that inhibition of cell proliferation by vitamin D in high-fat conditions limits vascular regeneration and recovery from peripheral ischemia in obesity.

Seno-destructive smooth muscle cells in the ascending aorta of patients with bicuspid aortic valve disease.

BACKGROUND: Ascending aortic aneurysms constitute an important hazard for individuals with a bicuspid aortic valve (BAV). However, the processes that degrade the aortic wall in BAV disease remain poorly understood. METHODS: We undertook in situ analysis of ascending aortas from 68 patients, seeking potentially damaging cellular senescence cascades. Aortas were assessed for senescence-associated-ß-galactosidase activity, p16Ink4a and p21 expression, and double-strand DNA breaks. The senescence-associated secretory phenotype (SASP) of cultured-aged BAV aortic smooth muscle cells (SMCs) was evaluated by transcript profiling and consequences probed by combined immunofluorescence and circular polarization microscopy. The contribution of p38 MAPK signaling was assessed by immunostaining and blocking strategies. FINDINGS: We uncovered SMCs at varying depths of cellular senescence within BAV- and tricuspid aortic valve (TAV)-associated aortic aneurysms. Senescent SMCs were also abundant in non-aneurysmal BAV aortas but not in non-aneurysmal TAV aortas. Multivariable analysis revealed that BAV disease independently associated with SMC senescence. Furthermore, SMC senescence was heightened at the convexity of aortas associated with right-left coronary cusp fusion. Aged BAV SMCs had a pronounced collagenolytic SASP. Moreover, senescent SMCs in the aortic wall were enriched with surface-localized MMP1 and surrounded by weakly birefringent collagen fibrils. The senescent-collagenolytic SMC phenotype depended on p38 MAPK signaling, which was chronically activated in BAV aortas. INTERPRETATION: We have identified a cellular senescence-collagen destruction axis in at-risk ascending aortas. This novel "seno-destructive" SMC phenotype could open new opportunities for managing BAV aortopathy. FUND: Canadian Institutes of Health Research, Lawson Health Research Institute, Heart and Stroke Foundation of Ontario/Barnett-Ivey Chair.

Wilms' tumor 1-associating protein regulates the proliferation of vascular smooth muscle cells.

Smooth muscle cells (SMCs) are called on to proliferate during vascular restructuring but must return to a nonproliferative state if remodeling is to appropriately terminate. To identify mediators of the reacquisition of replicative quiescence, we undertook gene expression screening in a uniquely plastic human SMC line. As proliferating SMCs shifted to a contractile and nonproliferative state, expression of TIMP-3, Axl, and KIAA0098 decreased whereas expression of complement C1s, cathepsin B, cellular repressor of E1A-activated genes increased. Wilms' tumor 1-associating protein (WTAP), a nuclear constituent of unknown function, was also upregulated as SMCs became nonproliferative. Furthermore, WTAP in the intima of injured arteries was substantially upregulated in the late stages of repair. Introduction of WTAP complementary DNA into human SMCs inhibited their proliferation, with a corresponding decrease in DNA synthesis and an increase in apoptosis. Knocking down endogenous WTAP increased SMC proliferation, because of increased DNA synthesis and G(1)/S phase transition, together with reduced apoptosis. WTAP was found to associate with the Wilms' tumor-1 protein in human SMCs and WTAP overexpression inhibited the binding of WT1 to an oligonucleotide containing a consensus WT1 binding site, whereas WTAP knockdown accentuated this interaction. Expression of the WT1 target genes, amphiregulin and Bcl-2, was suppressed in WTAP-overexpressing SMCs and increased in WTAP-deficient SMCs. Moreover, exogenous amphiregulin rescued the antiproliferative effect of WTAP. These findings identify WTAP as a novel regulator of the cell cycle and cell survival and implicate a WTAP-WT1 axis as a novel pathway for controlling vascular SMC phenotype.

Hepatic lipase expression in macrophages contributes to atherosclerosis in apoE-deficient and LCAT-transgenic mice.

Hepatic lipase (HL) has a well-established role in lipoprotein metabolism. However, its role in atherosclerosis is poorly understood. Here we demonstrate that HL deficiency raises the proatherogenic apoB-containing lipoprotein levels in plasma but reduces atherosclerosis in lecithin cholesterol acyltransferase (LCAT) transgenic (Tg) mice, similar to results previously observed with HL-deficient apoE-KO mice. These findings suggest that HL has functions that modify atherogenic risk that are separate from its role in lipoprotein metabolism. We used bone marrow transplantation (BMT) to generate apoE-KO and apoE-KO x HL-KO mice, as well as LCAT-Tg and LCAT-Tg x HL-KO mice, chimeric for macrophage HL gene expression. Using in situ RNA hybridization, we demonstrated localized production of HL by donor macrophages in the artery wall. We found that expression of HL by macrophages enhances early aortic lesion formation in both apoE-KO and LCAT-Tg mice, without changing the plasma lipid profile, lipoprotein lipid composition, or HL and lipoprotein lipase activities. HL does, however, enhance oxidized LDL uptake by peritoneal macrophages. These combined data demonstrate that macrophage-derived HL significantly contributes to early aortic lesion formation in two independent mouse models and identify a novel mechanism, separable from the role of HL in plasma lipoprotein metabolism, by which HL modulates atherogenic risk in vivo.

Pre-B-cell colony-enhancing factor regulates NAD+-dependent protein deacetylase activity and promotes vascular smooth muscle cell maturation.

Conversion of vascular smooth muscle cells (SMCs) from a proliferative state to a nonproliferative, contractile state confers vasomotor function to developing and remodeling blood vessels. Using a maturation-competent human SMC line, we determined that this shift in phenotype was accompanied by upregulation of pre-B-cell colony-enhancing factor (PBEF), a protein proposed to be a cytokine. Knockdown of endogenous PBEF increased SMC apoptosis and reduced the capacity of synthetic SMCs to mature to a contractile state. In keeping with these findings, human SMCs transduced with the PBEF gene had enhanced survival, an elongated bipolar morphology, and increased levels of h-caldesmon, smoothelin-A, smoothelin-B, and metavinculin. Notwithstanding some prior reports, PBEF did not have attributes of a cytokine but instead imparted the cell with increased nicotinamide phosphoribosyltransferase activity. Intracellular nicotinamide adenine dinucleotide (NAD+) content was increased in PBEF-overexpressing SMCs and decreased in PBEF-knockdown SMCs. Furthermore, NAD+-dependent protein deacetylase activity was found to be essential for SMC maturation and was increased by PBEF. Xenotransplantation of human SMCs into immunodeficient mice revealed an increased capacity for PBEF-overexpressing SMCs to mature and intimately invest nascent endothelial channels. This microvessel chimerism and maturation process was perturbed when SMC PBEF expression was lowered. These findings identify PBEF as a regulator of NAD+-dependent reactions in SMCs, reactions that promote, among other potential processes, the acquisition of a mature SMC phenotype.

Concurrent and Sustained Delivery of FGF2 and FGF9 from Electrospun Poly(ester amide) Fibrous Mats for Therapeutic Angiogenesis.

Therapeutic angiogenesis has emerged as a potential strategy to treat ischemic vascular diseases. However, systemic or local administration of growth factors is usually inefficient for maintaining the effective concentration at the site of interest due to their rapid clearance or degradation. In this study, we report a differential and sustained release of an angiogenic factor, fibroblast growth factor-2 (FGF2), and an arteriogenic factor, fibroblast growth factor-9 (FGF9), from α-amino acid-derived biodegradable poly(ester amide) (PEA) fibers toward targeting neovessel formation and maturation. FGF2 and FGF9 were dual loaded using a mixed blend and emulsion electrospinning technique and exhibited differential and sustained release from PEA fibers over 28 days with preserved bioactivity. In vitro angiogenesis assays showed enhanced endothelial cell (EC) tube formation and directed migration of smooth muscle cells (SMCs) to platelet-derived growth factor (PDGF)-BB and stabilized EC/SMC tube formation. FGF2/FGF9-loaded PEA fibers did not induce inflammatory responses in vitro using human monocytes or in vivo after their subcutaneous implantation into mice. Histological examination showed that FGF2/FGF9-loaded fibers induced cell niche recruitment around the site of implantation. Furthermore, controlled in vivo delivery of FGF9 to mouse tibialis anterior (TA) muscle resulted in a dose-dependent expansion of mesenchymal progenitor-like cell layers and extracellular matrix deposition. Our data suggest that the release of FGF2 and FGF9 from PEA fibers offers an efficient differential and sustained growth factor delivery strategy with relevance to therapeutic angiogenesis.

Niacin promotes revascularization and recovery of limb function in diet-induced obese mice with peripheral ischemia.

Niacin can reduce vascular disease risk in individuals with metabolic syndrome, but in light of recent large randomized controlled trials outcomes, its biological actions and clinical utility remain controversial. Niacin can improve endothelial function, vascular inflammation, and vascular regeneration, independent of correcting dyslipidemia, in various lean rodent models of vascular injury. Here, we tested whether niacin could directly improve endothelial cell angiogenic function during combined exposure to excess fatty acids and hypoxia, and whether intervention with niacin during continued feeding of western diet could improve revascularization and functional recovery in obese, hyperlipidemic mice with peripheral ischemia. Treatment with niacin (10 µmol/L) increased human microvascular endothelial cell angiogenic function during exposure to high fatty acids and hypoxia (2% oxygen), as determined by tube formation on Matrigel. To assess revascularization in vivo, we used western diet-induced obese mice with unilateral hind limb femoral artery ligation and excision. Treatment for 14 days postinjury with once daily i.p. injections of a low dose of niacin (50 mg/kg) improved recovery of hind limb use, in association with enhanced revascularization and decreased inflammation of the tibialis anterior muscle. These effects were concomitant with decreased plasma triglycerides, but not increased plasma apoAI. Thus, niacin improves endothelial tube formation under lipotoxic and hypoxic conditions, and moreover, promotes revascularization and functional hind limb recovery following ischemic injury in diet-induced obese mice with hyperlipidemia. These data may have implications for niacin therapy in the treatment of peripheral ischemic vascular disease associated with metabolic syndrome.