James M. Dominguez

Long-term type 1 diabetes influences haematopoietic stem cells by reducing vascular repair potential and increasing inflammatory monocyte generation in a murine model

Aims/hypothesisWe sought to determine the impact of long-standing type 1 diabetes on haematopoietic stem/progenitor cell (HSC) number and function and to examine the impact of modulating glycoprotein (GP)130 receptor in these cells.MethodsWild-type, gp130−/− and GFP chimeric mice were treated with streptozotocin to induce type 1 diabetes. Bone marrow (BM)-derived cells were used for colony-formation assay, quantification of side population (SP) cells, examination of gene expression, nitric oxide measurement and migration studies. Endothelial progenitor cells (EPCs), a population of vascular precursors derived from HSCs, were compared in diabetic and control mice. Cytokines were measured in BM supernatant fractions by ELISA and protein array. Flow cytometry was performed on enzymatically dissociated retina from gfp+ chimeric mice and used to assess BM cell recruitment to the retina, kidney and blood.ResultsBM cells from the 12-month-diabetic mice showed reduced colony-forming ability, depletion of SP-HSCs with a proportional increase in SP-HSCs residing in hypoxic regions of BM, decreased EPC numbers, and reduced eNos (also known as Nos3) but increased iNos (also known as Nos2) and oxidative stress-related genes. BM supernatant fraction showed increased cytokines, GP130 ligands and monocyte/macrophage stimulating factor. Retina, kidney and peripheral blood showed increased numbers of CD11b+/CD45hi/ CCR2+/Ly6Chi inflammatory monocytes. Diabetic gp130−/− mice were protected from development of diabetes-induced changes in their HSCs.Conclusions/interpretationThe BM microenvironment of type 1 diabetic mice can lead to changes in haematopoiesis, with generation of more monocytes and fewer EPCs contributing to development of microvascular complications. Inhibition of GP130 activation may serve as a therapeutic strategy to improve the key aspects of this dysfunction.

CNS Inflammation and Bone Marrow Neuropathy in Type 1 Diabetes

By using pseudorabies virus expressing green fluorescence protein, we found that efferent bone marrow-neural connections trace to sympathetic centers of the central nervous system in normal mice. However, this was markedly reduced in type 1 diabetes, suggesting a significant loss of bone marrow innervation. This loss of innervation was associated with a change in hematopoiesis toward generation of more monocytes and an altered diurnal release of monocytes in rodents and patients with type 1 diabetes. In the hypothalamus and granular insular cortex of mice with type 1 diabetes, bone marrow-derived microglia/macrophages were activated and found at a greater density than in controls. Infiltration of CD45(+)/CCR2(+)/GR-1(+)/Iba-1(+) bone marrow-derived monocytes into the hypothalamus could be mitigated by treatment with minocycline, an anti-inflammatory agent capable of crossing the blood-brain barrier. Our studies suggest that targeting central inflammation may facilitate management of microvascular complications.

Increased nitric oxide-mediated vasodilation of bone resistance arteries is associated with increased trabecular bone volume after endurance training in rats

Old age-associated osteoporosis is related to diminished bone blood flow and impaired nitric oxide (NO)-mediated vasodilation of the bone vasculature. Endurance exercise training restores the age-associated reduction of vasodilation in numerous vascular beds, as well as improving bone properties. The purpose of this study was to determine whether functional improvements in the bone vasculature are associated with increased bone properties after an endurance training intervention. Young adult (4-6 months) and old (24-26 months) male Fischer-344 rats remained sedentary or were trained (15 m/min walking, 15 degrees incline, 5 days/week, 10-12 weeks). Endothelium-dependent vasodilation of the femoral principal nutrient artery (PNA) was assessed in vitro using acetylcholine (ACh) and inhibitors of NO synthase (NOS) and cyclooxygenase (COX). PNA endothelium-dependent vasodilation was greater after training by 16% in young and by 24% in old animals. The NOS-mediated contribution to endothelium-dependent vasodilation was enhanced by 77% after training in old rats. Distal femur trabecular bone volume (BV/TV, %) was lower with old age in sedentary animals (young: 27+/-2%, old: 23+/-1%; P<0.05). Exercise-induced elevations in bone and marrow blood flow and the NOS signaling pathway were associated with greater BV/TV (young trained: 34+/-2%, old trained: 26+/-1%; P<0.05) relative to sedentary groups. These data demonstrate that training-induced increases in bone properties are associated with enhanced endothelium-dependent vasodilation through a NOS signaling pathway in the bone vasculature.

Aging and muscle fiber type alter K+ channel contributions to the myogenic response in skeletal muscle arterioles

Aging diminishes myogenic tone in arterioles from skeletal muscle. Recent evidence indicates that both large-conductance Ca2+-activated (BKCa) and voltage-dependent (KV) K+ channels mediate negative feedback control of the myogenic response. Thus we tested the hypothesis that aging increases the contributions of KV and BKCa channels to myogenic regulation of vascular tone. Because myogenic responsiveness differs between oxidative and glycolytic muscles, we predicted that KV and BKCa channel contributions to myogenic responsiveness vary with fiber type. Myogenic responses of first-order arterioles from the gastrocnemius and soleus muscles of 4- and 24-mo-old Fischer 344 rats were evaluated in the presence and absence of 4-aminopyridine (5 mM) or iberiotoxin (30 nM), inhibitors of KV and BKCa, respectively. 4-Aminopyridine enhanced myogenic tone with aging and normalized age-related differences in both muscle types. By contrast, iberiotoxin eliminated age-related differences in soleus arterioles and had no effect in gastrocnemius vessels. KV1.5 is an integral component of KV channels in vascular smooth muscle; therefore, we determined the relative protein expression of KV1.5, as well as BKCa, in soleus and gastrocnemius arterioles. Immunoblot analysis revealed no differences in KV1.5 protein with aging or between variant fiber types, whereas BKCa protein levels declined with age in arterioles from both muscle groups. Collectively, these results suggest that the contribution of BKCa to myogenic regulation of vascular tone changes with age in soleus muscle arterioles, whereas increased KV channel expression and negative feedback regulation of myogenic tone increases with advancing age in arterioles from both oxidative and glycolytic muscles.

Effects of spaceflight and ground recovery on mesenteric artery and vein constrictor properties in mice

Following exposure to microgravity, there is a reduced ability of astronauts to augment peripheral vascular resistance, often resulting in orthostatic hypotension. The purpose of this study was to test the hypothesis that mesenteric arteries and veins will exhibit diminished vasoconstrictor responses after spaceflight. Mesenteric arteries and veins from female mice flown on the Space Transportation System (STS)‐131 (n=11), STS‐133 (n=6), and STS‐135 (n=3) shuttle missions and respective ground‐based control mice (n=30) were isolated for in vitro experimentation. Vasoconstrictor responses were evoked in arteries via norepinephrine (NE), potassium chloride (KCl), and caffeine, and in veins through NE across a range of intraluminal pressures (2–12 cmH2O). Vasoconstriction to NE was also determined in mesenteric arteries at 1, 5, and 7 d postlanding. In arteries, maximal constriction to NE, KCl, and caffeine were reduced immediately following spaceflight and 1 d postflight. Spaceflight also reduced arterial ryanodine receptor‐3 mRNA levels. In mesenteric veins, there was diminished constriction to NE after flight. The results indicate that the impaired vasoconstriction following spaceflight occurs through the ryanodine receptor‐mediated intracellular Ca2+ release mechanism. Such vascular changes in astronauts could compromise the maintenance of arterial pressure during orthostatic stress.—Behnke, B. J., Stabley, J. N., McCullough, D. J., Davis, R. T., III, Dominguez, J. M., II, Muller‐Delp, J. M., Delp, M. D. Effects of spaceflight and ground recovery on mesenteric artery and vein constrictor properties in mice. FASEB J. 27, 399–409 (2013). www.fasebj.org

Spaceflight reduces vasoconstrictor responsiveness of skeletal muscle resistance arteries in mice

Cardiovascular adaptations to microgravity undermine the physiological capacity to respond to orthostatic challenges upon return to terrestrial gravity. The purpose of the present study was to investigate the influence of spaceflight on vasoconstrictor and myogenic contractile properties of mouse gastrocnemius muscle resistance arteries. We hypothesized that vasoconstrictor responses acting through adrenergic receptors [norepinephrine (NE)], voltage-gated Ca(2+) channels (KCl), and stretch-activated (myogenic) mechanisms would be diminished following spaceflight. Feed arteries were isolated from gastrocnemius muscles, cannulated on glass micropipettes, and physiologically pressurized for in vitro experimentation. Vasoconstrictor responses to intraluminal pressure changes (0-140 cmH(2)O), KCl (10-100 mM), and NE (10(-9)-10(-4) M) were measured in spaceflown (SF; n = 11) and ground control (GC; n = 11) female C57BL/6 mice. Spaceflight reduced vasoconstrictor responses to KCl and NE; myogenic vasoconstriction was unaffected. The diminished vasoconstrictor responses were associated with lower ryanodine receptor-2 (RyR-2) and ryanodine receptor-3 (RyR-3) mRNA expression, with no difference in sarcoplasmic/endoplasmic Ca(2+) ATPase 2 mRNA expression. Vessel wall thickness and maximal intraluminal diameter were unaffected by spaceflight. The data indicate a deficit in intracellular calcium release via RyR-2 and RyR-3 in smooth muscle cells as the mechanism of reduced contractile activity in skeletal muscle after spaceflight. Furthermore, the results suggest that impaired end-organ vasoconstrictor responsiveness of skeletal muscle resistance arteries contributes to lower peripheral vascular resistance and less tolerance of orthostatic stress in humans after spaceflight.

Effects of aging and exercise training on spinotrapezius muscle microvascular Po2 dynamics and vasomotor control

With advancing age, there is a reduction in exercise tolerance, resulting, in part, from a perturbed ability to match O(2) delivery to uptake within skeletal muscle. In the spinotrapezius muscle (which is not recruited during incline treadmill running) of aged rats, we tested the hypotheses that exercise training will 1) improve the matching of O(2) delivery to O(2) uptake, evidenced through improved microvascular Po(2) (Pm(O(2))), at rest and throughout the contractions transient; and 2) enhance endothelium-dependent vasodilation in first-order arterioles. Young (Y, ∼6 mo) and aged (O, >24 mo) Fischer 344 rats were assigned to control sedentary (YSED; n = 16, and OSED; n = 15) or exercise-trained (YET; n = 14, and OET; n = 13) groups. Spinotrapezius blood flow (via radiolabeled microspheres) was measured at rest and during exercise. Phosphorescence quenching was used to quantify Pm(O(2)) in vivo at rest and across the rest-to-twitch contraction (1 Hz, 5 min) transition in the spinotrapezius muscle. In a follow-up study, vasomotor responses to endothelium-dependent (acetylcholine) and -independent (sodium nitroprusside) stimuli were investigated in vitro. Blood flow to the spinotrapezius did not increase above resting values during exercise in either young or aged groups. Exercise training increased the precontraction baseline Pm(O(2)) (OET 37.5 ± 3.9 vs. OSED 24.7 ± 3.6 Torr, P < 0.05); the end-contracting Pm(O(2)) and the time-delay before Pm(O(2)) fell in the aged group but did not affect these values in the young. Exercise training improved maximal vasodilation in aged rats to acetylcholine (OET 62 ± 16 vs. OSED 27 ± 16%) and to sodium nitroprusside in both young and aged rats. Endurance training of aged rats enhances the Pm(O(2)) in a nonrecruited skeletal muscle and is associated with improved vascular smooth muscle function. These data support the notion that improvements in vascular function with exercise training are not isolated to the recruited muscle.

Aging and estrogen status: a possible endothelium-dependent vascular coupling mechanism in bone remodeling.

Bone loss with aging and menopause may be linked to vascular endothelial dysfunction. The purpose of the study was to determine whether putative modifications in endothelium-dependent vasodilation of the principal nutrient artery (PNA) of the femur are associated with changes in trabecular bone volume (BV/TV) with altered estrogen status in young (6 mon) and old (24 mon) female Fischer-344 rats. Animals were divided into 6 groups: 1) young intact, 2) old intact, 3) young ovariectomized (OVX), 4) old OVX, 5) young OVX plus estrogen replacement (OVX+E2), and 6) old OVX+E2. PNA endothelium-dependent vasodilation was assessed in vitro using acetylcholine. Trabecular bone volume of the distal femoral metaphysis was determined by microCT. In young rats, vasodilation was diminished by OVX and restored with estrogen replacement (intact, 82±7; OVX, 61±9; OVX+E2, 90±4%), which corresponded with similar modifications in BV/TV (intact, 28.7±1.6; OVX, 16.3±0.9; OVX+E2, 25.7±1.4%). In old animals, vasodilation was unaffected by OVX but enhanced with estrogen replacement (intact, 55±8; OVX, 59±7; OVX+E2, 92±4%). Likewise, modifications in BV/TV followed the same pattern (intact, 33.1±1.6; OVX, 34.4±3.7; OVX+E2, 42.4±2.1%). Furthermore, in old animals with low endogenous estrogen (i.e., intact and old OVX), vasodilation was correlated with BV/TV (R2 = 0.630; P<0.001). These data demonstrate parallel effects of estrogen on vascular endothelial function and BV/TV, and provide for a possible coupling mechanism linking endothelium-dependent vasodilation to bone remodeling.

Dicer Expression Exhibits a Tissue-Specific Diurnal Pattern That Is Lost during Aging and in Diabetes

Dysregulation of circadian rhythmicity is identified as a key factor in disease pathogenesis. Circadian rhythmicity is controlled at both a transcriptional and post-transcriptional level suggesting the role of microRNA (miRNA) and double-stranded RNA (dsRNA) in this process. Endonuclease Dicer controls miRNA and dsRNA processing, however the role of Dicer in circadian regulation is not known. Here we demonstrate robust diurnal oscillations of Dicer expression in central and peripheral clock control systems including suprachiasmatic nucleolus (SCN), retina, liver, and bone marrow (BM). The Dicer oscillations were either reduced or phase shifted with aging and Type 2 diabetes. The decrease and phase shift of Dicer expression was associated with a similar decrease and phase shift of miRNAs 146a and 125a-5p and with an increase in toxic Alu RNA. Restoring Dicer levels and the diurnal patterns of Dicer-controlled miRNA and RNA expression may provide new therapeutic strategies for metabolic disease and aging-associated complications.

Retinal Thickness Normative Data in Wild-Type Mice Using Customized Miniature SD-OCT

Objective To report normative data for retinal thickness in wild-type C57BL/6 mouse utilizing a miniature SD-OCT system. Methods Thirty adult mice (range: 3–5 months) were anesthetized and secured into the Bioptigen Spectral Domain Ophthalmic Imaging System. Right eye SD-OCT images were standardized by centralizing the optic nerve head (ONH) prior to image acquisition. Global and quadrant total retinal thickness (TRT) values were measured from retinal nerve fiber layer to retinal pigment epithelial layer. Posterior segment analyses also included the outer retinal layer (ORL) and inner retinal layer (IRL). Further sublayer analyses of four layers from the ORL and three layers comprising the IRL were also performed. Results The overall mean±SD global TRT in a C57BL/6 mouse model was 204.41±5.19 µm. Quadrant mean TRT values were 204.85±5.81 µm inferiorly, 204.97±6.71 µm nasally, 205.08±5.44 µm temporally, and 202.74±4.85 µm superiorly. Mean±SD thickness for ORL, and IRL were 126.37±10.01 µm, and 107.03±10.98 µm respectively. The mean±SD estimates for the four layers of the ORL were 18.23±2.73 µm, 26.04±4.21 µm, 63.8±6.23 µm, and 19.22±4.34 µm. Mean±SD values for the three IRL sublayers were 27.82±4.04 µm, 59.62±6.66 µm and 19.12±3.71 µm. Conclusion This study established normative values for the total retinal thickness and sublayer thickness for the wild-type C57BL/6 mice. Moreover, it provides a standard of retinal morphology, in a commonly used animal model, for evaluating therapeutic interventions and retinal disease pathophysiology.