Leroy L. Cooper

Impact of aging on the regenerative properties of bone marrow-, muscle-, and adipose-derived mesenchymal stem/stromal cells.

Mesenchymal stem/stromal cells (MSCs) are promising cell sources for regenerative therapies due to their multipotency and ready availability, but their application can be complicated by patient-specific factors like age or illness. MSCs have been investigated for the treatment of many musculoskeletal disorders, including osteoarthritis and osteoporosis. Due to the prevalence of these diseases in older populations, researchers have studied how aging affects MSC properties and have found that proliferation and differentiation potential are impaired. However, these effects have never been compared among MSCs isolated from multiple tissue sources in the same, healthy donor. Revealing differences in how MSCs are affected by age could help identify an optimal cell source for musculoskeletal therapies targeting older patients. MSCs were isolated from young and old rabbit bone marrow, muscle, and adipose tissue. Cell yield and viability were quantified after isolation procedures, and expansion properties were assessed using assays for proliferation, senescence, and colony formation. Multipotency was also examined using lineage-specific stains and spectrophotometry of metabolites. Results were compared between age groups and among MSC sources. Results showed that MSCs are differentially influenced by aging, with bone marrow-derived stem cells having impaired proliferation, senescence, and chondrogenic response, whereas muscle-derived stem cells and adipose-derived stem cells exhibited no negative effects. While age reduced overall cell yield and adipogenic potential of all MSC populations, osteogenesis and clonogenicity remained unchanged. These findings indicate the importance of age as a factor when designing cell-based therapies for older patients.

Cerebrovascular Damage Mediates Relations Between Aortic Stiffness and Memory

Aortic stiffness is associated with cognitive decline. Here, we examined the association between carotid-femoral pulse wave velocity and cognitive function and investigated whether cerebrovascular remodeling and parenchymal small vessel disease damage mediate the relation. Analyses were based on 1820 (60% women) participants in the Age, Gene/Environment Susceptibility—Reykjavik Study. Multivariable linear regression models adjusted for vascular and demographic confounders showed that higher carotid-femoral pulse wave velocity was related to lower memory score (standardized &bgr;: −0.071±0.023; P=0.002). Cerebrovascular resistance and white matter hyperintensities were each associated with carotid-femoral pulse wave velocity and memory (P<0.05). Together, cerebrovascular resistance and white matter hyperintensities (total indirect effect: −0.029; 95% CI, −0.043 to −0.017) attenuated the direct relation between carotid-femoral pulse wave velocity and memory (direct effect: −0.042; 95% CI, −0.087 to 0.003; P=0.07) and explained ≈41% of the observed effect. Our results suggest that in older adults, associations between aortic stiffness and memory are mediated by pathways that include cerebral microvascular remodeling and microvascular parenchymal damage.

Redox modification of ryanodine receptors by mitochondria-derived reactive oxygen species contributes to aberrant Ca2+ handling in ageing rabbit hearts

•  Ageing is associated with increased risk of sudden cardiac death due to malignant arrhythmias. •  Shortened refractoriness of Ca2+ release due to increased activity of Ca2+ release channels (RyRs) is recognized as an important contributor to cardiac‐triggered arrhythmias. However, molecular mechanisms of RyR dysfunction and its contribution to arrhythmias in ageing remain to be examined. •  Using ventricular myocytes isolated from old rabbit hearts we demonstrate that age‐associated increase in rate of production of reactive oxygen species (ROS) by mitochondria leads to the thiol‐oxidation of RyRs, which underlies the hyperactivity of the channels and thus shortened refractoriness of Ca2+ release in cardiomyocytes from the ageing heart. Mitochondria‐specific scavenging of ROS in old myocytes restored the redox status of RyRs, reducing SR Ca2+ leak and arrhythmogenic spontaneous Ca2+ waves. •  We conclude that increased ROS production by mitochondria contributes to age‐associated increased risk of stress‐induced arrhythmia and sudden cardiac death through thiol‐modifications of RyRs.

Components of hemodynamic load and cardiovascular events: the Framingham Heart Study.

Background— Elevated blood pressure is the leading modifiable risk factor for cardiovascular disease (CVD) and premature death. The blood pressure waveform consists of discrete hemodynamic components, derived from measured central pressure and flow, which may contribute separately to risk for an adverse outcome. However, pressure-flow measures have not been studied in a large, community-based sample. Methods and Results— We used proportional hazards models to examine the association of incident CVD with forward pressure wave amplitude, mean arterial pressure, and global reflection coefficient derived from wave separation analysis and echocardiography in 2492 participants (mean age 66±9 years, 56% women) in the Framingham Heart Study. During follow-up (0.04–6.8 years), 149 participants (6%) had a CVD event. In multivariable models adjusting for age, sex, antihypertensive therapy, body mass index, heart rate, total and high-density lipoprotein cholesterol concentrations, smoking, and the presence of diabetes mellitus, forward pressure wave amplitude (hazard ratio, 1.40; 95% confidence interval, 1.16–1.67; P=0.0003) was associated with incident CVD, whereas mean arterial pressure (hazard ratio, 1.10; 95% confidence interval, 0.94–1.29; P=0.25) and global wave reflection (hazard ratio, 0.93; 95% confidence interval, 0.78–1.12; P=0.58) were not. After adding systolic blood pressure and carotid-femoral pulse wave velocity to the model, forward pressure wave amplitude persisted as a correlate of events (hazard ratio, 1.33; 95% confidence interval, 1.05–1.68; P=0.02). Conclusions— Higher forward pressure wave amplitude (a measure of proximal aortic geometry and stiffness) was associated with increased risk for incident CVD, whereas mean arterial pressure and relative wave reflection (correlates of resistance vessel structure and function) were not associated with increased risk for incident CVD.

Hyperphosphorylation of RyRs Underlies Triggered Activity in Transgenic Rabbit Model of LQT2 Syndrome

Rationale: Loss-of-function mutations in human ether go-go (HERG) potassium channels underlie long QT syndrome type 2 (LQT2) and are associated with fatal ventricular tachyarrhythmia. Previously, most studies focused on plasma membrane–related pathways involved in arrhythmogenesis in long QT syndrome, whereas proarrhythmic changes in intracellular Ca2+ handling remained unexplored. Objective: We investigated the remodeling of Ca2+ homeostasis in ventricular cardiomyocytes derived from transgenic rabbit model of LQT2 to determine whether these changes contribute to triggered activity in the form of early after depolarizations (EADs). Methods and Results: Confocal Ca2+ imaging revealed decrease in amplitude of Ca2+ transients and sarcoplasmic reticulum Ca2+ content in LQT2 myocytes. Experiments using sarcoplasmic reticulum–entrapped Ca2+ indicator demonstrated enhanced ryanodine receptor (RyR)–mediated sarcoplasmic reticulum Ca2+ leak in LQT2 cells. Western blot analyses showed increased phosphorylation of RyR in LQT2 myocytes versus controls. Coimmunoprecipitation experiments demonstrated loss of protein phosphatases type 1 and type 2 from the RyR complex. Stimulation of LQT2 cells with &bgr;-adrenergic agonist isoproterenol resulted in prolongation of the plateau of action potentials accompanied by aberrant Ca2+ releases and EADs, which were abolished by inhibition of Ca2+/calmodulin-dependent protein kinase type 2. Computer simulations showed that late aberrant Ca2+ releases caused by RyR hyperactivity promote EADs and underlie the enhanced triggered activity through increased forward mode of Na+/Ca2+ exchanger type 1. Conclusions: Hyperactive, hyperphosphorylated RyRs because of reduced local phosphatase activity enhance triggered activity in LQT2 syndrome. EADs are promoted by aberrant RyR-mediated Ca2+ releases that are present despite a reduction of sarcoplasmic reticulum content. Those releases increase forward mode Na+/Ca2+ exchanger type 1, thereby slowing repolarization and enabling L-type Ca2+ current reactivation.

Electromechanical and structural alterations in the aging rabbit heart and aorta

Aging increases the risk for arrhythmias and sudden cardiac death (SCD). We aimed at elucidating aging-related electrical, functional, and structural changes in the heart and vasculature that account for this heightened arrhythmogenic risk. Young (5-9 mo) and old (3.5-6 yr) female New Zealand White (NZW) rabbits were subjected to in vivo hemodynamic, electrophysiological, and echocardiographic studies as well as ex vivo optical mapping, high-field magnetic resonance imaging (MRI), and histochemical experiments. Aging increased aortic stiffness (baseline pulse wave velocity: young, 3.54 ± 0.36 vs. old, 4.35 ± 0.28 m/s, P < 0.002) and diastolic (end diastolic pressure-volume relations: 3.28 ± 0.5 vs. 4.95 ± 1.5 mmHg/ml, P < 0.05) and systolic (end systolic pressure-volume relations: 20.56 ± 4.2 vs. 33.14 ± 8.4 mmHg/ml, P < 0.01) myocardial elastances in old rabbits. Electrophysiological and optical mapping studies revealed age-related slowing of ventricular and His-Purkinje conduction (His-to-ventricle interval: 23 ± 2.5 vs. 31.9 ± 2.9 ms, P < 0.0001), altered conduction anisotropy, and a greater inducibility of ventricular fibrillation (VF, 3/12 vs. 7/9, P < 0.05) in old rabbits. Histochemical studies confirmed an aging-related increased fibrosis in the ventricles. MRI showed a deterioration of the free-running Purkinje fiber network in ventricular and septal walls in old hearts as well as aging-related alterations of the myofibrillar orientation and myocardial sheet structure that may account for this slowed conduction velocity. Aging leads to parallel stiffening of the aorta and the heart, including an increase in systolic stiffness and contractility and diastolic stiffness. Increasingly, anisotropic conduction velocity due to fibrosis and altered myofibrillar orientation and myocardial sheet structure may contribute to the pathogenesis of VF in old hearts. The aging rabbit model represents a useful tool for elucidating age-related changes that predispose the aging heart to arrhythmias and SCD.

Aortic Stiffness, Cerebrovascular Dysfunction, and Memory

Background: Aortic stiffness is associated with cardiovascular and cerebrovascular events and cognitive decline. This mini-review focuses on relations of aortic stiffness with microvascular dysfunction and discusses the contribution of abnormal pulsatile hemodynamics to cerebrovascular damage and cognitive decline. We also provide a rationale for considering aortic stiffness as a putative and important contributor to memory impairment in older individuals. Summary: Aging is associated with stiffening of the aorta but not the muscular arteries, which reduces wave reflection and increases the transmission of pulsatility into the periphery. Aortic stiffening thereby impairs a protective mechanism that shields the peripheral microcirculation from excessive pulsatility within downstream target organs. Beyond midlife, aortic stiffness increases rapidly and exposes the cerebral microcirculation to abnormal pulsatile mechanical forces that are associated with microvascular damage and remodeling in the brain. Aortic stiffening and high-flow pulsatility are associated with alterations in the microvasculature of the brain; however, a mechanistic link between aortic stiffness and memory has not been established. We showed that in a community-based sample of older individuals, cerebrovascular resistance and white matter hyperintensities - markers of cerebrovascular remodeling and damage - mediated the relation between higher aortic stiffness and lower performance on memory function tests. These data suggest that microvascular and white matter damage associated with excessive aortic stiffness contribute to impaired memory function with advancing age. Key Messages: Increasing evidence suggests that vascular etiologies - including aortic stiffness and microvascular damage - contribute to memory impairment and the pathogenesis of dementia, including Alzheimers disease. Interventions that reduce aortic stiffness may delay memory decline among older individuals.

Relations of Arterial Stiffness With Postural Change in Mean Arterial Pressure in Middle-Aged Adults

Impaired regulation of blood pressure on standing can lead to adverse outcomes, including falls, syncope, and disorientation. Mean arterial pressure (MAP) typically increases on standing; however, an insufficient increase or a decline in MAP on standing may result in decreased cerebral perfusion. Orthostatic hypotension has been reported in older people with increased arterial stiffness, whereas the association between orthostatic change in MAP and arterial stiffness in young- to middle-aged individuals has not been examined. We analyzed orthostatic blood pressure response and comprehensive hemodynamic data in 3205 participants (1693 [53%] women) in the Framingham Heart Study Third Generation cohort. Participants were predominantly middle aged (mean age: 46±9 years). Arterial stiffness was assessed using carotid–femoral pulse wave velocity, forward pressure wave amplitude, and characteristic impedance of the aorta. Adjusting for standard cardiovascular disease risk factors, orthostatic change in MAP (6.9±7.7 mm Hg) was inversely associated with carotid–femoral pulse wave velocity (partial correlation, rp=−0.084; P<0.0001), forward wave amplitude (rp=−0.129; P<0.0001), and characteristic impedance (rp=−0.094; P<0.0001). The negative relation between forward wave amplitude and change in MAP on standing was accentuated in women (P=0.002 for sex interaction). Thus, higher aortic stiffness was associated with a blunted orthostatic increase in MAP, even in middle age. The clinical implications of these findings warrant further study.

Progesterone modulates SERCA2a expression and function in rabbit cardiomyocytes

We recently showed that progesterone treatment abolished arrhythmias and sudden cardiac death in a transgenic rabbit model of long QT syndrome type 2 (LQT2). Moreover, levels of cardiac sarco(endo)plasmic reticulum Ca(2+)-ATPase type 2a (SERCA2a) were upregulated in LQT2 heart extracts. We hypothesized that progesterone treatment upregulated SERCA2a expression, thereby reducing Ca(2+)-dependent arrhythmias in LQT2 rabbits. We therefore investigated the effect of progesterone on SERCA2a regulation in isolated cardiomyocytes. Cardiomyocytes from neonatal (3- to 5-day-old) rabbits were isolated, cultured, and treated with progesterone and other pharmacological agents. Immunoblotting was performed on total cell lysates and sarcoplasmic reticulum-enriched membrane fractions for protein abundance, and mRNA transcripts were quantified using real-time PCR. The effect of progesterone on baseline Ca(2+) transients and Ca(2+) clearance was determined using digital imaging. Progesterone treatment increased the total pool of SERCA2a protein by slowing its degradation. Using various pharmacological inhibitors of degradation pathways, we showed that progesterone-associated degradation of SERCA2a involves ubiquitination, and progesterone significantly decreases the levels of ubiquitin-tagged SERCA2a polypeptides. Our digital imaging data revealed that progesterone significantly shortened the decay and duration of Ca(2+) transients. Progesterone treatment increases protein levels and activity of SERCA2a. Progesterone stabilizes SERCA2a, in part, by decreasing the ubiquitination level of SERCA2a polypeptides.

Microvascular Function Contributes to the Relation Between Aortic Stiffness and Cardiovascular Events: The Framingham Heart Study.

Background—Arterial dysfunction contributes to cardiovascular disease (CVD) progression and clinical events. Inter-relations of aortic stiffness and vasodilator function with incident CVD remain incompletely studied. Methods and Results—We used proportional hazards models to relate individual measures of vascular function to incident CVD in 4547 participants (mean age, 51±11 years; 54% women) in 2 generations of Framingham Heart Study participants. During follow-up (0.02–13.83 years), 232 participants (5%) experienced new-onset CVD events. In multivariable models adjusted for cardiovascular risk factors, both higher carotid-femoral pulse wave velocity (hazard ratio [HR], 1.32; 95% confidence interval [CI], 1.07–1.63; P=0.01) and lower hyperemic mean flow velocity (HR, 0.84; 95% CI, 0.71–0.99; P=0.04) were associated significantly with incident CVD, whereas primary pressure wave amplitude (HR, 1.12; 95% CI, 0.99–1.27; P=0.06), baseline brachial diameter (HR, 1.09; 95% CI, 0.90–1.31; P=0.39), and flow-mediated vasodilation (HR, 0.85; 95% CI, 0.69–1.04; P=0.12) were not. In mediation analyses, 8% to 13% of the relation between aortic stiffness and CVD events was mediated by hyperemic mean flow velocity. Conclusions—Our results suggest that associations between aortic stiffness and CVD events are mediated by pathways that include microvascular damage and remodeling.