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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.

Description, The vasculature in ischemic muscle is rebuilt by the splitting of primordial vessels, led by VEGFR2-metastable endothelial cells., 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.

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-s-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.

Supplemental Digital Content is available in the text., 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.

Visual Abstract, Highlights • Short leukocyte telomeres have been associated with adverse cardiovascular outcomes in population studies, but this relationship has not translated to patient care. The authors report a telomere length autologous referencing strategy that has the potential to mark biological aging and to identify high-risk individuals. • Among 163 patients who underwent cardiovascular surgery, telomeres in leukocytes and skeletal muscle displayed age-related shortening, whereas the telomere length in the cardiac right atrium was stable during 6 decades of life. • The magnitude of the telomere length gap between cardiac atrial tissue and leukocytes was associated with post-operative complications and length of stay in the intensive care unit. • This study provided proof of concept that a single-time, internally referenced assessment of leukocyte telomere shortening behavior could inform acute risks in patients with cardiovascular disease., Summary Leukocyte telomere shortening reflects stress burdens and has been associated with cardiac events. However, the patient-specific clinical value of telomere assessment remains unknown. Moreover, telomere shortening cannot be inferred from a single telomere length assessment. The authors investigated and developed a novel strategy for gauging leukocyte telomere shortening using autologous cardiac atrial referencing. Using multitissue assessments from 163 patients who underwent cardiovascular surgery, we determined that the cardiac atrium-leukocyte telomere length difference predicted post-operative complexity. This constituted the first evidence that a single-time assessment of telomere dynamics might be salient to acute cardiac care.

Background Immunocompromised (IC) patients (pts) can have prolonged SARS-CoV-2 PCR positivity, even after resolution of COVID-19 symptoms. This study aimed to determine if viable virus could be detected in samples collected > 21 days after an initial positive (pos) SARS-CoV-2 PCR in IC pts. Methods We obtained 20 remnant SARS-CoV-2 PCR pos nasopharyngeal swabs from IC pts (bone marrow or solid organ transplant, high dose steroids, immunosuppressive medications) with a pos repeat PCR within the previous 30 days. The repeat specimens were cultured on Vero-hACE2-TMPRSS2 cells and incubated for 96 hours to assess viral viability. Viable RNA and infectious virus in the cultured cells were measured by qPCR and infectious plaque assays. RNA sequencing was performed on a HiSeq platform (Illumina). Samples also underwent SARS-CoV-2 antigen (Ag) testing (BD Veritor). Clinical data were extracted from the electronic health record by chart review. Results Pt characteristics are in Table 1. Viral cultures from the repeat specimen were negative (neg) for 18 pts and pos for 2 (Table 2). Pt 1 is a 60M treated with obinatuzumab 19 days prior to his first pos PCR test, with repeat specimen collected 21 days later (cycle threshold (Ct) not available). Pt 1 had a low viral titer (27 PFU/mL) & a D614G mutation on sequencing. Pt 2 is a 75M treated with rituximab 10 days prior to his first pos PCR test, with repeat specimen collected 23 days later (Ct 27.56/27.74). Pt 2 had a high viral titer (2e6 PFU/mL) and D614G, S98F, and S813I mutations. Demographics of Study Population (N=20) Characteristics of patients with a positive SARS-CoV-2 viral culture Conclusion 90% of specimens collected > 21 days after an initial pos SARS-CoV-2 PCR did not have viable virus detected on their repeat specimen. The 2 pts with pos viral cultures had active hematologic malignancies treated with an anti-CD20 mAb at the time of COVID-19 diagnosis. One pt had a high concentration of active, viable virus. No known variants of concern were noted in this cohort, collected in Q2 2020, though prolonged replication is a risk for variant development. Further data are needed about risk factors for persistent viable viral shedding & methods to prevent transmission of viable virus from IC hosts. Disclosures Victoria J. Fraser, MD, CDC Epicenters (Grant/Research Support)Cigna/Express Scripts (Other Financial or Material Support, Spouse is Chief Clinical Officer)Doris Duke Fund to Retain Clinical Scientists (Grant/Research Support, Research Grant or Support)Foundation for Barnes-Jewish Hospital (Grant/Research Support, Research Grant or Support)NIH (Grant/Research Support, Research Grant or Support) Victoria J. Fraser, MD, Centers for Disease Control and Prevention (Individual(s) Involved: Self): Grant/Research Support, Research Grant or Support; Cigna/Express Scripts (Individual(s) Involved: Spouse/Partner): Employee; Doris Duke Charitable Foundation (Individual(s) Involved: Self): Grant/Research Support, Research Grant or Support; National Institutes of Health (Individual(s) Involved: Self): Grant/Research Support, Research Grant or Support; The Foundation for Barnes-Jewish Hospital (Individual(s) Involved: Self): Grant/Research Support, Research Grant or Support Michael S. Diamond, MD, PhD, Carnival Corporation (Consultant)Emergent BioSolutions (Grant/Research Support)Fortress Biotech (Consultant)Immunome (Advisor or Review Panel member)Inbios (Consultant)Moderna (Grant/Research Support, Advisor or Review Panel member)Vir Biotechnology (Consultant, Grant/Research Support) Carey-Ann Burnham, PhD, BioFire (Grant/Research Support, Other Financial or Material Support)bioMerieux (Grant/Research Support)Cepheid (Consultant, Grant/Research Support)Luminex (Grant/Research Support)Roche (Other Financial or Material Support) Carey-Ann Burnham, PhD, BioFire (Individual(s) Involved: Self): Grant/Research Support; bioMerieux (Individual(s) Involved: Self): Grant/Research Support, Scientific Research Study Investigator, Speakers’ bureau; Cepheid (Individual(s) Involved: Self): Consultant, Grant/Research Support, Scientific Research Study Investigator; Luminex (Individual(s) Involved: Self): Scientific Research Study Investigator Hilary Babcock, MD, MPH, FIDSA, FSHEA, Nothing to disclose

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.

The two-spotted spider mite (TSSM), Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most polyphagous herbivores, feeding on more than 1,100 plant species. Its wide host range suggests that TSSM has an extraordinary ability to modulate its digestive and xenobiotic physiology. The analysis of the TSSM genome revealed the expansion of gene families that encode proteins involved in digestion and detoxification, many of which were associated with mite responses to host shifts. The majority of plant defense compounds that directly impact mite fitness are ingested. They interface mite compounds aimed at counteracting their effect in the gut. Despite several detailed ultrastructural studies, our knowledge of the TSSM digestive tract that is needed to support the functional analysis of digestive and detoxification physiology is lacking. Here, using a variety of histological and microscopy techniques, and a diversity of tracer dyes, we describe the organization and properties of the TSSM alimentary system. We define the cellular nature of floating vesicles in the midgut lumen that are proposed to be the site of intracellular digestion of plant macromolecules. In addition, by following the TSSM's ability to intake compounds of defined sizes, we determine a cut off size for the ingestible particles. Moreover, we demonstrate the existence of a distinct filtering function between midgut compartments which enables separation of molecules by size. Furthermore, we broadly define the spatial distribution of the expression domains of genes involved in digestion and detoxification. Finally, we discuss the relative simplicity of the spider mite digestive system in the context of mite's digestive and xenobiotic physiology, and consequences it has on the effectiveness of plant defenses.

Not so long ago, the traditional wisdom in medicine was that there is nothing you can do for a stroke. “Neurologists, when they were called about a stroke patient would say, ‘Make them comfortable, I’ll see them in the morning,’” Dr. Antoine Hakim, recipient of the 2017 Gairdner Wightman

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.

Collagen fibrils become resistant to cleavage over time. We hypothesized that resistance to type I collagen proteolysis not only marks biological aging but also drives it. To test this, we followed mice with a targeted mutation (Col1a1(r/r) ) that yields collagenase-resistant type I collagen. Compared with wild-type littermates, Col1a1(r/r) mice had a shortened lifespan and developed features of premature aging including kyphosis, weight loss, decreased bone mineral density, and hypertension. We also found that vascular smooth muscle cells (SMCs) in the aortic wall of Col1a1(r/r) mice were susceptible to stress-induced senescence, displaying senescence-associated ß-galactosidase (SA-ßGal) activity and upregulated p16(INK4A) in response to angiotensin II infusion. To elucidate the basis of this pro-aging effect, vascular SMCs from twelve patients undergoing coronary artery bypass surgery were cultured on collagen derived from Col1a1(r/r) or wild-type mice. This revealed that mutant collagen directly reduced replicative lifespan and increased stress-induced SA-ßGal activity, p16(INK4A) expression, and p21(CIP1) expression. The pro-senescence effect of mutant collagen was blocked by vitronectin, a ligand for αvß3 integrin that is presented by denatured but not native collagen. Moreover, inhibition of αvß3 with echistatin or with αvß3-blocking antibody increased senescence of SMCs on wild-type collagen. These findings reveal a novel aging cascade whereby resistance to collagen cleavage accelerates cellular aging. This interplay between extracellular and cellular compartments could hasten mammalian aging and the progression of aging-related diseases.

Summary Cell migration is central to tissue repair and regeneration but must proceed with precise directionality to be productive. Directional migration requires external cues but also depends on the extent to which cells can inherently maintain their direction of crawling. We report that the NAD+ biosynthetic enzyme, nicotinamide phosphoribosyltransferase (Nampt/PBEF/visfatin), mediates directionally persistent migration of vascular smooth muscle cells (SMCs). Time-lapse microscopy of human SMCs subjected to Nampt inhibition revealed chaotic motility whereas SMCs transduced with the Nampt gene displayed highly linear migration paths. Ordered motility conferred by Nampt was associated with downsizing of the lamellipodium, reduced lamellipodium wandering around the cell perimeter, and increased lamellipodial protrusion rates. These protrusive and polarity-stabilizing effects also enabled spreading SMCs to undergo bipolar elongation to an extent not typically observed in vitro. Nampt was found to localize to lamellipodia and fluorescence recovery of Nampt–eGFP after photobleaching revealed microtubule-dependent transport of Nampt to the leading edge. In addition, Nampt was found to associate with, and activate, Cdc42, and Nampt-driven directional persistence and lamellipodium anchoring required Cdc42. We conclude that high-fidelity SMC motility is coordinated by a Nampt–Cdc42 axis that yields protrusive but small and anchored lamellipodia. This novel, NAD+-synthesis-dependent control over motility may be crucial for efficient repair and regeneration of the vasculature, and possibly other tissues.

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.

Introduction: Ascertaining the biological age of patients with advanced vascular disease could advance risk assessment and management. The extent to which telomeres shorten in leukocytes could be a marker of biological age because it reflects the accumulation of replication stresses imposed on leukocyte progenitors. However, because of wide, genetic variability in leukocyte telomere length (TL), a single leukocyte TL measurement does not reliably indicate telomere shortening. Hypothesis: We hypothesized that the difference in length of telomeres in “non-replicating” muscle-rich tissue and that of circulating leukocytes provides a patient-specific index of telomere shortening in patients with advanced vascular disease. Methods: TL in leukocytes, skeletal muscle, and right atrial cardiac muscle were measured from 134 patients undergoing coronary or thoracic aortic surgery, using quantitative polymerase chain reaction. Relationships between leukocyte TL or the muscle-leukocyte TL difference (ΔTL) and early post-operative outcomes were tested using Cox proportional hazard and binary logistic regression analyses. Results: Telomeres in cardiac muscle and skeletal muscle were significantly longer than those in leukocytes (p Conclusions: Right atrium-leukocyte ΔTL provides an index of telomere shortening and may inform outcomes in patients with advanced vascular disease. This two-component telomere measurement may reflect the biological age of individuals with chronic vascular disease.

The therapeutic potential of angiogenic growth factors has not been realized. This may be because formation of endothelial sprouts is not followed by their muscularization into vasoreactive arteries. Using microarray expression analysis, we discovered that fibroblast growth factor 9 (FGF9) was highly upregulated as human vascular smooth muscle cells (SMCs) assemble into layered cords. FGF9 was not angiogenic when mixed with tissue implants or delivered to the ischemic mouse hind limb, but instead orchestrated wrapping of SMCs around neovessels. SMC wrapping in implants was driven by sonic hedgehog-mediated upregulation of PDGFRβ. Computed tomography microangiography and intravital microscopy revealed that microvessels formed in the presence of FGF9 had enhanced capacity to receive flow and were vasoreactive. Moreover, the vessels persisted beyond 1 year, remodeling into multilayered arteries paired with peripheral nerves. This mature physiological competency was attained by targeting mesenchymal cells rather than endothelial cells, a finding that could inform strategies for therapeutic angiogenesis and tissue engineering.

His protein visualization tools have impressed his colleagues, but it was Lewis Kay’s decision to share his methods with the scientific community that earned him the 2017 Canada Gairdner International Award. “He’s always been very, very good at sharing these techniques with other scientists

Extending the productive lifespan of human cells could have major implications for diseases of aging, such as atherosclerosis. We identified a relationship between aging of human vascular smooth muscle cells (SMCs) and nicotinamide phosphoribosyltransferase (Nampt/PBEF/Visfatin), the rate-limiting enzyme for NAD+ salvage from nicotinamide. Replicative senescence of SMCs was preceded by a marked decline in the expression and activity of Nampt. Furthermore, reducing Nampt activity with the antagonist FK866 induced premature senescence in SMCs, assessed by serial quantification of the proportion of cells with senescence-associated beta-galactosidase activity. In contrast, introducing the Nampt gene into aging human SMCs delayed senescence and substantially lengthened cell lifespan, together with enhanced resistance to oxidative stress. Nampt-mediated SMC lifespan extension was associated with increased activity of the NAD+-dependent longevity enzyme SIRT1 and was abrogated in Nampt-overexpressing cells transduced with a dominant-negative form of SIRT1 (H363Y). Nampt overexpression also reduced the fraction of p53 that was acetylated on lysine 382, a target of SIRT1, suppressed an age-related increase in p53 expression, and increased the rate of p53 degradation. Moreover, add-back of p53 with recombinant adenovirus blocked the anti-aging effects of Nampt. These data indicate that Nampt is a longevity protein that can add stress-resistant life to human SMCs by optimizing SIRT1-mediated p53 degradation.

It has been increasingly appreciated that aldosterone elicits acute vascular effects through nongenomic signaling pathways. Our previous studies demonstrated that aldosterone attenuated phenylephrine-mediated constriction in intact vessels [via phosphatidylinositol 3-kinase-dependent nitric oxide synthase activation] but enhanced vasoconstrictor responses in endothelium-denuded arteries. To determine the mechanism of this vasoconstrictor response, we assessed the effect of aldosterone on myosin light-chain phosphorylation and contraction in clonal adult human vascular smooth muscle cells. Acute aldosterone exposure mediated dose-dependent myosin light-chain phosphorylation, inhibited by spironolactone and phosphatidylinositol 3-kinase inhibition. These rapid effects of aldosterone were mimicked by estradiol and hydrocortisone and were also inhibitable by both spironolactone and eplerenone. In parallel to its effects on myosin light-chain phosphorylation, aldosterone mediated dose-dependent contraction responses that were inhibited by spironolactone. Comparable contractile responses were seen with both 17β-estradiol and hydrocortisone. In total, these data are consistent with a mechanism of acute aldosterone-mediated contraction common to both glucocorticoids and estrogen. Steroid-mediated vasoconstriction may represent an important pathobiological mechanism of vascular disease, especially in the setting of preexisting endothelial dysfunction.

Individuals with a bicuspid aortic valve (BAV) are at increased risk for ascending aortic dilation and dissection. Loss of aortic medial smooth muscle cells (SMCs) and disruption of the extracellular matrix are well-recognized pathologies, but the underlying cellular mechanisms remain elusive. We tested the hypothesis that the dilated aorta in patients with BAV was marked by accelerated cellular aging. Samples of human ascending aorta were obtained from individuals with BAV undergoing thoracic aorta replacement (n=37, age 54.7±2.2, aortic diameter 4.8±0.9 cm) or patients with a tricuspid aortic valve and non-dilated aorta undergoing heart transplantation or coronary bypass procedures (n=6, age 55.3±8.1, aortic diameter 3.1±0.3 cm). Assessment of fresh aortic samples for senescence-associated β-galactosidase revealed evidence for rare medial cell senescence that was 4.2-fold more prevalent in dilated aortas (0.83±0.10%) than in non-dilated aortas (0.20±0.10%, p=0.048). Expression of p16 was abundantly detected in medial SMCs within dilated aortas (27.0±2.1%) and 3-fold more abundant than in non-dilated aortas (8.9±1.8%, p

Normalizing the tumor vasculature has been proposed as a therapeutic paradigm. However, to function normally, microvessels must exist as a vasoreactive and hierarchical network with red blood cells flowing single file through capillaries. Such a network has not been identified in malignant tumors. We previously found that fibroblast growth factor 9 (FGF9) could stabilize new blood vessels in ischemic muscle. To determine if FGF9 impacted tumors vessels, renal carcinoma (Renca) cells, expressing GFP or FGF9, were implanted into the subcapsular space of female Balb/c mice. After 14 days, the resulting FGF9-tumors had 17% fewer microvessels than control tumors (p=0.003) but the vessels had a collagen-fortified basement membrane and were more extensively covered with pericytes (4-fold, p=0.015) and smooth muscle cells (14-fold, p=0.002). Notably, this was associated with reduced pulmonary metastases (p=0.029). Intravital video microscopy revealed that FGF9 converted a haphazard web of channels into a hierarchal network with arterioles, capillaries, and venules. There was also a 33% reduction in vessel length density (p=0.034), a 67% reduction in mean lumen diameter (p Conclusion: FGF9 can impart an otherwise dysfunctional tumor microvasculature with hierarchy, vasoreactivity, and improved oxygen delivery, via selective amplification of PDGFRß-expressing mesenchymal stromal cells. These findings suggest an approach to driving microvascular network differentiation, to an extent not observed previously, to pacify the tumor.

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 PDGFRβ-positive stromal cells in the tumor and blocking PDGFRβ 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.

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.

The cholesterol biosynthetic pathway produces numerous signaling molecules. Oxysterols through liver X receptor (LXR) activation regulate cholesterol efflux, whereas the non-sterol mevalonate metabolite, geranylgeranyl pyrophosphate (GGPP), was recently demonstrated to inhibit ABCA1 expression directly, through antagonism of LXR and indirectly through enhanced RhoA geranylgeranylation. We used HMG-CoA reductase inhibitors (statins) to test the hypothesis that reduced synthesis of mevalonate metabolites would enhance cholesterol efflux and attenuate foam cell formation. Preincubation of THP-1 macrophages with atorvastatin, dose dependently (1-10 microm) stimulated cholesterol efflux to apolipoprotein AI (apoAI, 10-60%, p < 0.05) and high density lipoprotein (HDL(3)) (2-50%, p < 0.05), despite a significant decrease in cholesterol synthesis (2-90%). Atorvastatin also increased ABCA1 and ABCG1 mRNA abundance (30 and 35%, p < 0.05). Addition of mevalonate, GGPP or farnesyl pyrophosphate completely blocked the statin-induced increase in ABCA1 expression and apoAI-mediated cholesterol efflux. A role for RhoA was established, because two inhibitors of Rho protein activity, a geranylgeranyl transferase inhibitor and C3 exoenzyme, increased cholesterol efflux to apoAI (20-35%, p < 0.05), and macrophage expression of dominant-negative RhoA enhanced cholesterol efflux to apoAI (20%, p < 0.05). In addition, atorvastatin increased the RhoA levels in the cytosol fraction and decreased the membrane localization of RhoA. Atorvastatin treatment activated peroxisome proliferator activated receptor gamma and increased LXR-mediated gene expression suggesting that atorvastatin induces cholesterol efflux through a molecular cascade involving inhibition of RhoA signaling, leading to increased peroxisome proliferator activated receptor gamma activity, enhanced LXR activation, increased ABCA1 expression, and cholesterol efflux. Finally, statin treatment inhibited cholesteryl ester accumulation in macrophages challenged with atherogenic hypertriglyceridemic very low density lipoproteins indicating that statins can regulate foam cell formation.

Plasma membrane protrusion is fundamental to cell motility, but its regulation by the extracellular environment is not well elucidated. We have quantified lamellipodial protrusion dynamics in human vascular smooth muscle cells exposed to fibroblast growth factor 2 (FGF-2) and type I collagen, two distinct ligands presented to vascular cells during arterial remodeling. Video microscopy revealed that FGF-2 stimulated a modest increase in lamellipodial protrusion rate that peaked within 15 min. This response was associated with immediate but transient activation of Rac1 and was inhibited in cells infected with retrovirus containing cDNA encoding dominant-negative Rac1. A 1-h exposure to FGF-2 also set up a second phase of more striking lamellipodial protrusion evident at 24–36 h. This delayed response was most pronounced when cells were on type 1 collagen and was associated with FGF-2-induced expression of collagenase-1 that localized to the edge of protruding lamellipodia. Moreover, late membrane protrusion was inhibited when cells were on collagenase-resistant type I collagen, implicating degraded collagen as a mediator. For cells on collagen, the immediate activation of Rac1 by FGF-2 was followed by a sustained wave of Rac1 activation that was inhibited when cleavage of the collagen triple helix was prevented and also by blockade of αvβ3 integrin. We conclude that lamellipodial protrusion in smooth muscle cells can be regulated by waves of Rac1 activation, corresponding to the sequential presentation of FGF-2 and remodeled collagen. The findings thus reveal a previously unrecognized level of coordination among extracellular input that enables cells to maintain protrusive activity over prolonged periods.

Background Foam cell formation by intimal smooth muscle cells (SMCs) inhibits the elaboration of extracellular matrix, which is detrimental to plaque stabilization. In the present study, we examined the lipoproteins and receptors involved in human SMC foam cell formation and investigated the ability of 24( S ),25‐epoxycholesterol [24( S ),25‐EC], an oxysterol agonist of the liver X receptor, to attenuate SMC foam cell formation. Methods and Results Incubation of human internal thoracic SMCs with atherogenic lipoproteins demonstrated that low‐density lipoprotein (LDL), but not oxidized or acetylated LDL, was the primary lipoprotein taken up, resulting in marked cholesteryl ester deposition (6‐fold vs 1.8‐fold; P (S) ,25‐EC attenuated LDL uptake (−90% and −47% respectively; P sterol regulatory element–binding protein‐2 expression (−30% and −17%, respectively; P LDL receptor expression (−75% and −40%, respectively; P myosin regulatory light chain interacting protein expression (7‐ and 3‐fold, respectively; P (S) ,25‐EC increased adenosine triphosphate–binding cassettes A1– and G1–mediated cholesterol efflux to apolipoprotein AI (1.9‐fold; P 3 (1.3‐fold; P (S) ,25‐EC, unlike a nonsteroidal liver X receptor agonist, T0901317, did not stimulate sterol regulatory element–binding protein‐1c –mediated fatty acid synthesis or triglyceride accumulation. 24( S ),25‐EC preserved the assembly of fibronectin and type I collagen by SMCs. Conclusions The oxysterol 24 (S) ,25‐EC prevented foam cell formation in human SMCs by attenuation of LDL receptor–mediated LDL uptake and stimulation of cholesterol efflux, restoring the elaboration of extracellular matrix. In contrast to T0901317, 24 (S) ,25‐EC prevented the development of a triglyceride‐rich foam cell phenotype. ( J Am Heart Assoc . 2012;1:e000810 doi : 10.1161/JAHA.112.000810 .)

Vascular smooth muscle cell (SMC) migration is characterized by extension of the lamellipodia at the leading edge, lamellipodial attachment to substrate, and release of the rear (uropod) of the cell, all of which enable forward movement. However, little is known regarding the role of intracellular cytosolic Ca2+concentration ([Ca2+]i) in coordinating these distinct activities of migrating SMCs. The objective of our study was to determine whether regional changes of Ca2+orchestrate the migratory cycle in human vascular SMCs. We carried out Ca2+imaging using digital fluorescence microscopy of fura-2 loaded human smooth muscle cells. We found that motile SMCs exhibited Ca2+waves that characteristically swept from the rear of polarized cells toward the leading edge. Ca2+waves were less evident in nonpolarized, stationary cells, although acute stimulation of these SMCs with the agonists platelet-derived growth factor-BB or histamine could elicit transient rise of [Ca2+]i. To investigate a role for Ca2+waves in the migratory cycle, we loaded cells with the Ca2+chelator BAPTA, which abolished Ca2+waves and significantly reduced retraction, supporting a causal role for Ca2+in initiation of retraction. However, lamellipod motility was still evident in BAPTA-loaded cells. The incidence of Ca2+oscillations was reduced when Ca2+release from intracellular stores was disrupted with the sarcoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin or by treatment with the inositol 1,4,5-trisphosphate receptor blocker 2-aminoethoxy-diphenyl borate or xestospongin C, implicating Ca2+stores in generation of waves. We conclude that Ca2+waves are essential for migration of human vascular SMCs and can encode cell polarity.

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. © 2011 American Society for Investigative Pathology.

A vital role of vascular smooth muscle cells (SMCs) is to stabilize the artery wall by elaborating fibrils of type I collagen. This is especially important in atherosclerotic lesions. However, SMCs in these lesions can be laden with lipids and the impact of this modification on collagen fibril formation is unknown. To address this, we converted human vascular SMCs to a foam cell state by incubating them with either LDL or VLDL. Biochemical markers of a SMC phenotype were preserved. However, microscopic tracking revealed a profound perturbation in the ability of the cells to assemble collagen fibrils, reducing assembly by up to 79%. This dysfunction was mirrored by an inability of smooth muscle foam cells to assemble fibronectin. Lipid-loaded SMCs did not display a generalized defect in the actin cytoskeleton and the formation of vinculin-containing focal adhesion complexes was preserved. However, lipid-loaded SMCs were unable to assemble fibrillar adhesion complexes and clustering of tensin and α5β1 integrin was disordered. Moreover, phosphorylation of tensin, required for fibrillar adhesion complex formation, was suppressed by up to 57%, with a concomitant decrease in activation of Src and FAK and restriction of activated Src to the cell edges. Forced activation of Src-FAK signaling in lipid-engorged SMCs rescued both fibrillar adhesion formation and fibrillogenesis. We conclude that lipid accumulation by SMCs disables the machinery for collagen and fibronectin assembly. This previously unknown relationship between atherogenic lipids and integrin-based signaling could underlie plaque vulnerability.

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.

Platelet-activating-Factor (PAF) and its structural analogues formed upon low density lipoprotein oxidation are involved in atherosclerotic plaque formation and may signal through PAF-receptor (PAF-R) expressed in human macrophages and in certain smooth muscle cells (SMCs) in the media, but rarely in the intima of human plaques. Our aim was to determine which SMC phenotype expresses PAF-R and whether this receptor is functional in cell migration. Circulating SMC progenitors and two phenotypically distinct clones of proliferative, epithelioid phenotype vs contractile, spindle-shaped SMCs from the media of adult internal thoracic artery were studied for the presence of PAF-receptor (PAF-R). The levels of specific mRNA were obtained by reverse transcription/real-time PCR, the protein expression was deduced from immunohistochemistry staining, and the functional transmigration assay was performed by Boyden chamber-type chemotaxis assay. Only SMCs of spindle-shape and synthetic phenotype expressed both mRNA and PAF-R protein and in the functional test migrated at low concentrations of PAF. Two unrelated, specific PAF-R antagonists inhibited PAF-induced migration, but did not modify the migration initiated by PDGF. The presence of functional PAF-R in arterial spindle-shaped SMCs of synthetic phenotype may be important for their migration from the media into the intima and atherosclerotic plaques formation.

To date, all cases of familial partial lipodystrophy type 2 (FPLD2; Mendelian Inheritance in Man 151660) result from missense mutations in LMNA, which encodes nuclear lamin A/C (Mendelian Inheritance in Man 150330).The objective of the study was to carry out mutational analysis of LMNA in two sisters with a particularly severe FPLD2 phenotype.This was a descriptive case report with molecular studies.The study was conducted at a referral center.We report two sisters of South Asian origin. The first presented with acanthosis nigricans at age 5 yr, diabetes with insulin resistance, hypertension and hypertriglyceridemia at age 13 yr, and partial lipodystrophy starting at puberty. Her sister and their mother had a similar metabolic profile and physical features, and their mother died of vascular disease at age 32 yr.There were no interventions.LMNA sequencing showed that the sisters were each heterozygous for a novel GC mutation at the intron 8 consensus splice donor site, which was absent from the genomes of 300 healthy individuals. The retention of intron 8 in mRNA predicted a prematurely truncated lamin A isoform (516 instead of 664 amino acids) with 20 nonsense 3'-terminal residues. The mutant lamin A isoform failed to interact normally with emerin and failed to localize to the nuclear envelope.This is the first LMNA splicing mutation to be associated with FPLD2, and it causes a severe clinical and metabolic phenotype.

Hutchinson-Gilford progeria syndrome (HGPS; MIM 176670) is a rare disease characterized by accelerated aging. In this study, light and immunofluorescence microscopy were used to assess morphological changes, measures of cell growth kinetics and gene expression profiles in HGPS cells and normal fibroblasts in culture. A filtering strategy was developed based on differentially expressed transcripts seen consistently across three culture stages based on cell passage number. This filtering strategy produced a list of 66 unique differentially expressed genes, of which approximately 40% were upregulated in HGPS cells compared to normal fibroblasts. The increased mRNA expression in HGPS cells that was seen for one gene defined using this strategy--namely ANK3--was validated using quantitative reverse-transcriptase amplification, Western analysis and immunofluorescence microscopy, all of which showed significantly increased ankyrin G expression. These findings demonstrate differences in morphology, growth kinetics and mRNA expression profiles in HGPS cells compared to normal fibroblasts in culture, including increased expression of ANK3/ankyrin G. Furthermore, other genes that co-clustered with ANK3 might provide mechanistic clues regarding senescence in cultured HGPS cells.

Plasma membrane protrusion is fundamental to cell motility, but its regulation by the extracellular environment is not well elucidated. We have quantified lamellipodial protrusion dynamics in human vascular smooth muscle cells exposed to fibroblast growth factor 2 (FGF-2) and type I collagen, two distinct ligands presented to vascular cells during arterial remodeling. Video microscopy revealed that FGF-2 stimulated a modest increase in lamellipodial protrusion rate that peaked within 15 min. This response was associated with immediate but transient activation of Rac1 and was inhibited in cells infected with retrovirus containing cDNA encoding dominant-negative Rac1. A 1-h exposure to FGF-2 also set up a second phase of more striking lamellipodial protrusion evident at 24-36 h. This delayed response was most pronounced when cells were on type 1 collagen and was associated with FGF-2-induced expression of collagenase-1 that localized to the edge of protruding lamellipodia. Moreover, late membrane protrusion was inhibited when cells were on collagenase-resistant type I collagen, implicating degraded collagen as a mediator. For cells on collagen, the immediate activation of Rac1 by FGF-2 was followed by a sustained wave of Rac1 activation that was inhibited when cleavage of the collagen triple helix was prevented and also by blockade of alpha(v)beta(3) integrin. We conclude that lamellipodial protrusion in smooth muscle cells can be regulated by waves of Rac1 activation, corresponding to the sequential presentation of FGF-2 and remodeled collagen. The findings thus reveal a previously unrecognized level of coordination among extracellular input that enables cells to maintain protrusive activity over prolonged periods.