Cardiac-Referenced Leukocyte Telomere Length and Outcomes After Cardiovascular Surgery.

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.

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.

Collagenase-resistant collagen promotes mouse aging and vascular cell senescence.

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.

Intrinsic directionality of migrating vascular smooth muscle cells is regulated by NAD(+) biosynthesis.

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.

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.

Coordinated integrin and growth factor regulation of primary keratinocyte migration mediated through extracellular signal regulated kinase and phosphoinositide 3-kinase.

We have examined coordinated integrin and growth factor regulation of primary keratinocyte migration mediated by phosphoinositide 3-kinase (PI3K) and mitogen-activated extracellular-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK). On collagen I and fibronectin substrates, both epidermal growth factor (EGF) and hepatocyte growth factor (HGF) stimulated chemokinetic (random) and chemotactic (directional) migration. On provisional matrix, a combination of fibronectin and fibrin found in the early phase of wound healing, EGF and HGF-stimulated significant chemotactic but little or no chemokinetic cell movement. Blocking mAbs to integrin alpha2beta1 and alpha5beta1 effectively inhibited EGF- and HGF-stimulated chemokinetic and chemotactic cell movement on collagen I and fibronectin, respectively; however, HGF-stimulated chemotactic migration on collagen I was only partially inhibited by alpha2beta1 blocking mAb. Differentiated keratinocytes underwent reduced chemokinetic and chemotactic migration compared with undifferentiated keratinocytes; however, EGF-stimulated migration was reduced more than HGF-stimulated migration. When the migratory response on collagen I and fibronectin was assessed in the presence of the MEK-specific inhibitor PD98059, EGF- and HGF-stimulated chemotaxis was significantly reduced, whereas PD98059 had little effect on the stimulated chemokinesis. PI3K-specific inhibitor LY294002 reduced EGF- and HGF-stimulated chemokinesis and chemotaxis on collagen I and fibronectin. Thus beta1 integrins acted in concert with EGF and HGF to regulate migration of primary keratinocytes on extracellular matrix components via PI3K and MEK/ERK.