Publications by authors named "Maribeth H Johnson"

78 Publications

The Glucocorticoid Receptor in Osterix-Expressing Cells Regulates Bone Mass, Bone Marrow Adipose Tissue, and Systemic Metabolism in Female Mice During Aging.

J Bone Miner Res 2021 Nov 8. Epub 2021 Nov 8.

Department of Cellular Biology and Anatomy, Augusta University, Augusta, GA, USA.

Hallmarks of aging-associated osteoporosis include bone loss, bone marrow adipose tissue (BMAT) expansion, and impaired osteoblast function. Endogenous glucocorticoid levels increase with age, and elevated glucocorticoid signaling, associated with chronic stress and dysregulated metabolism, can have a deleterious effect on bone mass. Canonical glucocorticoid signaling through the glucocorticoid receptor (GR) was recently investigated as a mediator of osteoporosis during the stress of chronic caloric restriction. To address the role of the GR in an aging-associated osteoporotic phenotype, the current study utilized female GR conditional knockout (GR-CKO; GR :Osx-Cre+) mice and control littermates on the C57BL/6 background aged to 21 months and studied in comparison to young (3- and 6-month-old) mice. GR deficiency in Osx-expressing cells led to low bone mass and BMAT accumulation that persisted with aging. Surprisingly, however, GR-CKO mice also exhibited alterations in muscle mass (reduced % lean mass and soleus fiber size), accompanied by reduced voluntary physical activity, and also exhibited higher whole-body metabolic rate and elevated blood pressure. Moreover, increased lipid storage was observed in GR-CKO osteoblastic cultures in a glucocorticoid-dependent fashion despite genetic deletion of the GR, and could be reversed via pharmacological inhibition of the mineralocorticoid receptor (MR). These findings provide evidence of a role for the GR (and possibly the MR) in facilitating healthy bone maintenance with aging in females. The effects of GR-deficient bone on whole-body physiology also demonstrate the importance of bone as an endocrine organ and suggest evidence for compensatory mechanisms that facilitate glucocorticoid signaling in the absence of osteoblastic GR function; these represent new avenues of research that may improve understanding of glucocorticoid signaling in bone toward the development of novel osteogenic agents. © 2021 American Society for Bone and Mineral Research (ASBMR).
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http://dx.doi.org/10.1002/jbmr.4468DOI Listing
November 2021

Age-associated changes in microRNAs affect the differentiation potential of human mesenchymal stem cells: Novel role of miR-29b-1-5p expression.

Bone 2021 12 14;153:116154. Epub 2021 Aug 14.

Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29403, United States of America; Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC 29403, United States of America; Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, Augusta, GA 30912, United States of America; Center for Healthy Aging, Medical College of Georgia, Augusta University, Augusta, GA 30912, United States of America. Electronic address:

Age-associated osteoporosis is widely accepted as involving the disruption of osteogenic stem cell populations and their functioning. Maintenance of the local bone marrow (BM) microenvironment is critical for regulating proliferation and differentiation of the multipotent BM mesenchymal stromal/stem cell (BMSC) population with age. The potential role of microRNAs (miRNAs) in modulating BMSCs and the BM microenvironment has recently gained attention. However, miRNAs expressed in rapidly isolated BMSCs that are naïve to the non-physiologic standard tissue culture conditions and reflect a more accurate in vivo profile have not yet been reported. Here we directly isolated CD271 positive (+) BMSCs within hours from human surgical BM aspirates without culturing and performed microarray analysis to identify the age-associated changes in BMSC miRNA expression. One hundred and two miRNAs showed differential expression with aging. Target prediction and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the up-regulated miRNAs targeting genes in bone development pathways were considerably enriched. Among the differentially up-regulated miRNAs the novel passenger strand miR-29b-1-5p was abundantly expressed as a mature functional miRNA with aging. This suggests a critical arm-switching mechanism regulates the expression of the miR-29b-1-5p/3p pair shifting the normally degraded arm, miR-29b-1-5p, to be the dominantly expressed miRNA of the pair in aging. The normal guide strand miR-29b-1-3p is known to act as a pro-osteogenic miRNA. On the other hand, overexpression of the passenger strand miR-29b-1-5p in culture-expanded CD271+ BMSCs significantly down-regulated the expression of stromal cell-derived factor 1 (CXCL12)/ C-X-C chemokine receptor type 4 (SDF-1(CXCL12)/CXCR4) axis and other osteogenic genes including bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor 2 (RUNX2). In contrast, blocking of miR-29b-1-5p function using an antagomir inhibitor up-regulated expression of BMP-2 and RUNX2 genes. Functional assays confirmed that miR-29b-1-5p negatively regulates BMSC osteogenesis in vitro. These novel findings provide evidence of a pathogenic anti-osteogenic role for miR-29b-1-5p and other miRNAs in age-related defects in osteogenesis and bone regeneration.
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http://dx.doi.org/10.1016/j.bone.2021.116154DOI Listing
December 2021

Indoleamine 2,3 dioxygenase, age, and immune activation in people living with HIV.

J Investig Med 2021 08 19;69(6):1238-1244. Epub 2021 Apr 19.

Institute of Cellular Medicine, Faculty of Medical Science, Newcastle University, Newcastle upon Tyne, UK.

Immune activation complicates HIV despite antiretroviral therapy (ART). Indoleamine 2,3 dioxygenase (IDO) catabolizes tryptophan (T) to kynurenine (K), regulating immune activity, and IDO activity increases with age. This study examines the relationship of IDO activity, bacterial translocation, and aging in people living with HIV (PLWH) on ART. Samples and data from PLWH on ART from the Centers for AIDS Research Network of Integrated Clinical Systems and from matched HIV-uninfected patients (controls) from the Multicenter AIDS Cohort Study and the Women's Interagency HIV Study were analyzed. The ratio of K to T (K:T) and neopterin were indicators of inflammation; 16S ribosomal DNA (16S rDNA) and lipopolysaccharide (LPS) were markers of bacterial translocation. Samples and data from 205 PLWH and 99 controls were analyzed. PLWH had higher K:T values across all ages, with a significant relationship between age and K:T for both groups. CD4 count or CD4 nadir had no association with K:T. There was no positive association between level of 16S rDNA or LPS detection and K:T. K:T and neopterin were associated. PLWH had elevated IDO activity, at younger ages, despite ART. This study suggests K:T ratio increases with age in both groups and is elevated in PLWH at all ages compared with age-matched controls.
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http://dx.doi.org/10.1136/jim-2021-001794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8319090PMC
August 2021

Stroke promotes the development of brain atrophy and delayed cell death in hypertensive rats.

Sci Rep 2020 11 19;10(1):20233. Epub 2020 Nov 19.

Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, 914 New Baillie Street, HM Building Room 116, Augusta, GA, 30901, USA.

Post-stroke cognitive impairment (PSCI) is a major source of disability, affecting up to two thirds of stroke survivors with no available therapeutic options. The condition remains understudied in preclinical models due to its delayed presentation. Although hypertension is a leading risk factor for dementia, how ischemic stroke contributes to this neurodegenerative condition is unknown. In this study, we used a model of hypertension to study the development of PSCI and its mechanisms. Spontaneously hypertensive rats (SHR) were compared to normotensive rats and were subjected to 1-h middle cerebral artery occlusion or sham surgery. Novel object recognition, passive avoidance test and Morris water maze were used to assess cognition. In addition, brain magnetic resonance images were obtained 12-weeks post-stroke and tissue was collected for immunohistochemistry and protein quantification. Stroked animals developed impairment in long-term memory at 4-weeks post-stroke despite recovery from motor deficits, with hypertensive animals showing some symptoms of anhedonia. Stroked SHRs displayed grey matter atrophy and had a two-fold increase in apoptosis in the ischemic borderzone and increased markers of inflammatory cell death and DNA damage at 12 weeks post-stroke. This indicates that preexisting hypertension exacerbates the development of secondary neurodegeneration after stroke beyond its acute effects on neurovascular injury.
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http://dx.doi.org/10.1038/s41598-020-75450-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7678843PMC
November 2020

Deferoxamine Treatment Prevents Post-Stroke Vasoregression and Neurovascular Unit Remodeling Leading to Improved Functional Outcomes in Type 2 Male Diabetic Rats: Role of Endothelial Ferroptosis.

Transl Stroke Res 2021 08 1;12(4):615-630. Epub 2020 Sep 1.

Ralph H. Johnson VA Medical Center, Charleston, SC, USA.

It is a clinically well-established fact that patients with diabetes have very poor stroke outcomes. Yet, the underlying mechanisms remain largely unknown. Our previous studies showed that male diabetic animals show greater hemorrhagic transformation (HT), profound loss of cerebral vasculature in the recovery period, and poor sensorimotor and cognitive outcomes after ischemic stroke. This study aimed to determine the impact of iron chelation with deferoxamine (DFX) on (1) cerebral vascularization patterns and (2) functional outcomes after stroke in control and diabetic rats. After 8 weeks of type 2 diabetes induced by a combination of high-fat diet and low-dose streptozotocin, male control and diabetic animals were subjected to thromboembolic middle cerebral artery occlusion (MCAO) and randomized to vehicle, DFX, or tPA/DFX and followed for 14 days with behavioral tests. Vascular indices (vascular volume and surface area), neurovascular remodeling (AQP4 polarity), and microglia activation were measured. Brain microvascular endothelial cells (BMVEC) from control and diabetic animals were evaluated for the impact of DFX on ferroptotic cell death. DFX treatment prevented vasoregression and microglia activation while improving AQP4 polarity as well as blood-brain barrier permeability by day 14 in diabetic rats. These pathological changes were associated with improvement of functional outcomes. In control rats, DFX did not have an effect. Iron increased markers of ferroptosis and lipid reactive oxygen species (ROS) to a greater extent in BMVECs from diabetic animals, and this was prevented by DFX. These results strongly suggest that (1) HT impacts post-stroke vascularization patterns and recovery responses in diabetes, (2) treatment of bleeding with iron chelation has differential effects on outcomes in comorbid disease conditions, and (3) iron chelation and possibly inhibition of ferroptosis may provide a novel disease-modifying therapeutic strategy in the prevention of post-stroke cognitive impairment in diabetes.
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http://dx.doi.org/10.1007/s12975-020-00844-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917163PMC
August 2021

Microglia knockdown reduces inflammation and preserves cognition in diabetic animals after experimental stroke.

J Neuroinflammation 2020 Apr 28;17(1):137. Epub 2020 Apr 28.

Program in Clinical and Experimental Therapeutics, University of Georgia College of Pharmacy, Augusta, GA, USA.

Introduction: Unfortunately, over 40% of stroke victims have pre-existing diabetes which not only increases their risk of stroke up to 2-6 fold, but also worsens both functional recovery and the severity of cognitive impairment. Our lab has recently linked the chronic inflammation in diabetes to poor functional outcomes and exacerbated cognitive impairment, also known as post-stroke cognitive impairment (PSCI). Although we have shown that the development of PSCI in diabetes is associated with the upregulation and the activation of pro-inflammatory microglia, we have not established direct causation between the two. To this end, we evaluated the role of microglia in the development of PSCI.

Methods: At 13 weeks of age, diabetic animals received bilateral intracerebroventricular (ICV) injections of short hairpin RNA (shRNA) lentiviral particles targeting the colony stimulating factor 1 receptor (CSF1R). After 14 days, animals were subjected to 60 min middle cerebral artery occlusion (MCAO) or sham surgery. Adhesive removal task (ART), novel object recognition (NOR), and 2-trial Y-maze were utilized to evaluate sensorimotor and cognitive function. Tissue from freshly harvested brains was analyzed by flow cytometry and immunohistochemistry.

Results: CSF1R silencing resulted in a 94% knockdown of residential microglia to relieve inflammation and improve myelination of white matter in the brain. This prevented cognitive decline in diabetic animals.

Conclusion: Microglial activation after stroke in diabetes may be causally related to the development of delayed neurodegeneration and PSCI.
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http://dx.doi.org/10.1186/s12974-020-01815-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7189436PMC
April 2020

Kynurenine suppresses osteoblastic cell energetics in vitro and osteoblast numbers in vivo.

Exp Gerontol 2020 02 17;130:110818. Epub 2019 Dec 17.

Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, Augusta, GA, USA; Department of Orthopaedic Surgery, Augusta University, Augusta, GA, USA. Electronic address:

Aging is a progressive process associated with declining tissue function over time. Kynurenine, an oxidized metabolite of the essential amino acid tryptophan that increases in abundance with age, drives cellular processes of aging and dysfunction in many tissues, and recent work has focused on understanding the pathways involved in the harmful effects of kynurenine on bone. In this study, we sought to investigate the effects of controlled kynurenine administration on osteoblast bioenergetics, in vivo osteoblast abundance, and marrow fat accumulation. Additionally, as an extension of earlier studies with dietary administration of kynurenine, we investigated the effects of kynurenine on Hdac3 and NCoR1 expression and enzymatic deacetylase activity as potential mechanistic contributors to the effects of kynurenine on osteoblasts. Kynurenine administration suppressed cellular metabolism in osteoblasts at least in part through impaired mitochondrial respiration, and suppressed osteoblastic numbers in vivo with no concurrent effects on marrow adiposity. Deleterious effects of kynurenine treatment on osteoblasts were more pronounced in female models as compared to males. However, kynurenine treatment did not inhibit Hdac3's enzymatic deacetylase activity nor its repression of downstream glucocorticoid signaling. As such, future work will be necessary to determine the mechanisms by which increased kynurenine contributes to aging bone bioenergetics. The current study provides novel further support for the idea that kynurenine contributes to impaired osteoblastic function, and suggests that impaired matrix production by kynurenine-affected osteoblasts is attributed in part to impaired osteoblastic bioenergetics. As circulating kynurenine levels in increase with age, and human bone density inversely correlates with the serum kynurenine to tryptophan ratio, these mechanisms may have important relevance in the etiology and pathogenesis of osteoporosis in humans.
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http://dx.doi.org/10.1016/j.exger.2019.110818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003726PMC
February 2020

Delayed Administration of Angiotensin II Type 2 Receptor (AT2R) Agonist Compound 21 Prevents the Development of Post-stroke Cognitive Impairment in Diabetes Through the Modulation of Microglia Polarization.

Transl Stroke Res 2020 08 3;11(4):762-775. Epub 2019 Dec 3.

Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Ave. MSC, Charleston, SC, 908, USA.

A disabling consequence of stroke is cognitive impairment, occurring in 12%-48% of patients, for which there is no therapy. A critical barrier is the lack of understanding of how post-stroke cognitive impairment (PSCI) develops. While 70% of stroke victims present with comorbid diseases such as diabetes and hypertension, the limited use of comorbid disease models in preclinical research further contributes to this lack of progress. To this end, we used a translational model of diabetes to study the development of PSCI. In addition, we evaluated the application of compound 21 (C21), an angiotensin II Type 2 receptor agonist, for the treatment of PSCI by blinding the treatment assignment, setting strict inclusion criteria, and implementing a delayed administration time point. Diabetes was induced by a high-fat diet (HFD) and low-dose streptozotocin (STZ) combination. Control and diabetic rats were subjected to 1 h middle cerebral artery occlusion (MCAO) or sham surgery. Adhesive removal task (ART) and two-trial Y-maze were utilized to test sensorimotor and cognitive function. Three days post-stroke, rats that met the inclusion criteria were administered C21 or vehicle in drinking water at a dose of 0.12 mg/kg/day for 8 weeks. Samples from freshly harvested brains were analyzed by flow cytometry and immunohistochemistry (IHC). Diabetes exacerbated the development of PSCI and increased inflammation and demyelination. Delayed administration of C21 3 days post-stroke reduced mortality and improved sensorimotor and cognitive deficits. It also reduced inflammation and demyelination through modulation of the M1:M2 ratio in the diabetic animals.
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http://dx.doi.org/10.1007/s12975-019-00752-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7266715PMC
August 2020

Decreased pericellular matrix production and selection for enhanced cell membrane repair may impair osteocyte responses to mechanical loading in the aging skeleton.

Aging Cell 2020 01 19;19(1):e13056. Epub 2019 Nov 19.

Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, Augusta, GA.

Transient plasma membrane disruptions (PMD) occur in osteocytes with in vitro and in vivo loading, initiating mechanotransduction. The goal here was to determine whether osteocyte PMD formation or repair is affected by aging. Osteocytes from old (24 months) mice developed fewer PMD (-76% females, -54% males) from fluid shear than young (3 months) mice, and old mice developed fewer osteocyte PMD (-51%) during treadmill running. This was due at least in part to decreased pericellular matrix production, as studies revealed that pericellular matrix is integral to formation of osteocyte PMD, and aged osteocytes produced less pericellular matrix (-55%). Surprisingly, osteocyte PMD repair rate was faster (+25% females, +26% males) in osteocytes from old mice, and calcium wave propagation to adjacent nonwounded osteocytes was blunted, consistent with impaired mechanotransduction downstream of PMD in osteocytes with fast PMD repair in previous studies. Inducing PMD via fluid flow in young osteocytes in the presence of oxidative stress decreased postwounding cell survival and promoted accelerated PMD repair in surviving cells, suggesting selective loss of slower-repairing osteocytes. Therefore, as oxidative stress increases during aging, slower-repairing osteocytes may be unable to successfully repair PMD, leading to slower-repairing osteocyte death in favor of faster-repairing osteocyte survival. Since PMD are an important initiator of mechanotransduction, age-related decreases in pericellular matrix and loss of slower-repairing osteocytes may impair the ability of bone to properly respond to mechanical loading with bone formation. These data suggest that PMD formation and repair mechanisms represent new targets for improving bone mechanosensitivity with aging.
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http://dx.doi.org/10.1111/acel.13056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6974724PMC
January 2020

The glucocorticoid receptor in osteoprogenitors regulates bone mass and marrow fat.

J Endocrinol 2019 07 1. Epub 2019 Jul 1.

M McGee-Lawrence, Cellular Biology and Anatomy, Augusta University, Augusta, United States.

Excess fat within bone marrow is associated with lower bone density. Metabolic stressors such as chronic caloric restriction (CR) can exacerbate marrow adiposity, and increased glucocorticoid signaling and adrenergic signaling are implicated in this phenotype. The current study tested the role of glucocorticoid signaling in CR-induced stress by conditionally deleting the glucocorticoid receptor (GR) in bone marrow osteoprogenitors (Osx1-Cre) of mice subjected to CR and ad libitum diets. Conditional knockout of the GR (GR-CKO) reduced cortical and trabecular bone mass as compared to wildtype (WT) mice under both ad libitum and CR conditions. No interaction was detected between genotype and diet, suggesting that the GR is not required for CR-induced skeletal changes. The lower bone mass in GR-CKO mice, and the further suppression of bone by CR, resulted from suppressed bone formation. Interestingly, treatment with the -adrenergic receptor antagonist propranolol mildly but selectively improved metrics of cortical bone mass in GR-CKO mice during CR, suggesting interaction between adrenergic and glucocorticoid signaling pathways that affects cortical bone. GR-CKO mice dramatically increased marrow fat under both ad libitum and CR-fed conditions, and surprisingly propranolol treatment was unable to rescue CR-induced marrow fat in either WT or GR-CKO mice. Additionally, serum corticosterone levels were selectively elevated in GR-CKO mice with CR, suggesting the possibility of bone-hypothalamus-pituitary-adrenal crosstalk during metabolic stress. This work highlights the complexities of glucocorticoid and β-adrenergic signaling in stress-induced changes in bone mass, and the importance of GR function in suppressing marrow adipogenesis while maintaining healthy bone mass.
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http://dx.doi.org/10.1530/JOE-19-0230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938567PMC
July 2019

Monomethylfumarate protects against ovariectomy-related changes in body composition.

J Endocrinol 2019 07 1. Epub 2019 Jul 1.

C Isales, Neuroscience and Regenerative Medicine, Augusta University, Augusta, 30912, United States.

Osteoporosis, low bone mass that increases fracture susceptibility, affects approximately 75 million individuals in the United States, Europe and Japan, with the number of osteoporotic fractures expected to increase by more than 3-fold over the next 50 years. Bone mass declines with age, although the mechanisms for this decrease are unclear. Aging enhances production of reactive oxygen species, which can affect bone formation and breakdown. The multiple sclerosis drug Tecfidera contains dimethylfumarate, which is rapidly metabolized to monomethylfumarate (MMF); MMF is thought to function through nuclear factor erythroid-derived-2-like-2 (Nrf2), a transcription factor activated by oxidative stress which induces the expression of endogenous anti-oxidant systems. We hypothesized that MMF-elicited increases in anti-oxidants would inhibit osteopenia induced by ovariectomy, as a model of aging-related osteoporosis and high oxidative stress. We demonstrated that MMF activated Nrf2 and induced anti-oxidant Nrf2 target gene expression in bone marrow-derived mesenchymal stem cells. Sham-operated or ovariectomized adult female mice were fed chow with or without MMF and various parameters monitored. Ovariectomy produced the expected effects, decreasing bone mineral density and increasing body weight, fat mass, bone marrow adiposity and serum receptor activator of nuclear factor-kappa-B ligand (RANKL) levels. MMF decreased fat but not lean mass. MMF improved trabecular bone microarchitecture after adjustment for body weight, although the unadjusted data showed few differences; MMF also tended to increase adjusted cortical bone and to reduce bone marrow adiposity and serum RANKL levels. Because these results suggest the possibility that MMF might be beneficial for bone, further investigation seems warranted.
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http://dx.doi.org/10.1530/JOE-18-0691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938560PMC
July 2019

Inhibition of Osteocyte Membrane Repair Activity via Dietary Vitamin E Deprivation Impairs Osteocyte Survival.

Calcif Tissue Int 2019 02 24;104(2):224-234. Epub 2018 Oct 24.

Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, 1120 15th St, Augusta, GA, 30912, USA.

Osteocytes experience plasma membrane disruptions (PMD) that initiate mechanotransduction both in vitro and in vivo in response to mechanical loading, suggesting that osteocytes use PMD to sense and adapt to mechanical stimuli. PMD repair is crucial for cell survival; antioxidants (e.g., alpha-tocopherol, also known as Vitamin E) promote repair while reactive oxygen species (ROS), which can accumulate during exercise, inhibit repair. The goal of this study was to determine whether depleting Vitamin E in the diet would impact osteocyte survival and bone adaptation with loading. Male CD-1 mice (3 weeks old) were fed either a regular diet (RD) or Vitamin E-deficient diet (VEDD) for up to 11 weeks. Mice from each dietary group either served as sedentary controls with normal cage activity, or were subjected to treadmill exercise (one bout of exercise or daily exercise for 5 weeks). VEDD-fed mice showed more PMD-affected osteocytes (+ 50%) after a single exercise bout suggesting impaired PMD repair following Vitamin E deprivation. After 5 weeks of daily exercise, VEDD mice failed to show an exercise-induced increase in osteocyte PMD formation, and showed signs of increased osteocytic oxidative stress and impaired osteocyte survival. Surprisingly, exercise-induced increases in cortical bone formation rate were only significant for VEDD-fed mice. This result may be consistent with previous studies in skeletal muscle, where myocyte PMD repair failure (e.g., with muscular dystrophy) initially triggers hypertrophy but later leads to widespread degeneration. In vitro, mechanically wounded MLO-Y4 cells displayed increased post-wounding necrosis (+ 40-fold) in the presence of HO, which could be prevented by Vitamin E pre-treatment. Taken together, our data support the idea that antioxidant-influenced osteocyte membrane repair is a vital aspect of bone mechanosensation in the osteocytic control of PMD-driven bone adaptation.
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http://dx.doi.org/10.1007/s00223-018-0487-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452877PMC
February 2019

Soy Phosphatidylglycerol Reduces Inflammation in a Contact Irritant Ear Edema Mouse Model In Vivo.

J Pharmacol Exp Ther 2018 07 25;366(1):1-8. Epub 2018 Apr 25.

Charlie Norwood VA Medical Center, Augusta, Georgia (V.C., W.B.B.); Institute of Molecular Medicine and Genetics, Department of Medicine (D.X., M.S., W.B.B.), Department of Physiology (D.X., V.C., M.S., A.W., A.C.E., K.A.B., W.B.B.), Department of Family Medicine (D.X.), Department of Neuroscience and Regenerative Medicine (M.H.J.), and Division of Dermatology, Department of Medicine (W.B.B.), Medical College of Georgia, Augusta University, Augusta, Georgia; and Apeliotus Technologies, Inc., Philadelphia, Pennsylvania (J.G.E., W.B.B.)

We have previously shown that phosphatidylglycerol (PG) regulates the function of keratinocytes, the predominant cells that compose the epidermis, inhibiting the proliferation of rapidly dividing keratinocytes. In particular, soy PG, a PG mixture with a high proportion of polyunsaturated fatty acids, is efficacious at inhibiting these proliferating keratinocytes. Psoriasis is a skin disorder characterized by hyperproliferation of keratinocytes and inflammation. Data in the lung suggest that PG in pulmonary surfactant inhibits inflammation. To investigate the possibility of using PG containing polyunsaturated fatty acids for the treatment of psoriasis, we examined the effect of soy PG on inflammation induced by the application of 12--tetradecanoylphorbol 13-acetate (TPA), a contact irritant, to mouse ears in vivo. We monitored ear thickness and weight as a measure of ear edema, as well as CD45-positive immune cell infiltration. Our results indicate that soy PG when applied together with 1,25-dihydroxyvitamin D (vitamin D), an agent known to acutely disrupt the skin barrier, suppressed ear edema and inhibited the infiltration of CD45-positive immune cells. On the other hand, neither PG nor vitamin D alone was effective. The combination also decreased tumor necrosis factor- (TNF) levels. This result suggested the possibility that PG was not permeating the skin barrier efficiently. Therefore, in a further study we applied PG in a penetration-enhancing vehicle and found that it inhibited inflammation induced by the phorbol ester and decreased CD45-positive immune cell infiltration. Our results suggest the possibility of using soy PG as a topical treatment option for psoriasis.
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http://dx.doi.org/10.1124/jpet.117.244756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5988020PMC
July 2018

Protein kinase D1 conditional null mice show minimal bone loss following ovariectomy.

Mol Cell Endocrinol 2018 10 9;474:176-183. Epub 2018 Mar 9.

Institute for Regenerative and Reparative Medicine, Augusta University, Augusta, GA 30912, United States; Department of Orthopaedic Surgery, Augusta University, Augusta, GA 30912, United States; Department of Medicine, Augusta University, Augusta, GA 30912, United States; Department of Neuroscience and Regenerative Medicine, Augusta University, Augusta, GA 30912, United States.

We previously found that 3- and 6-month-old male mice with conditional ablation of protein kinase D1 (PRKD1) in osteoprogenitor cells (expressing Osterix) exhibited reduced bone mass. Others have demonstrated similar effects in young female PRKD1-deficient mice. Here we examined the bone resorptive response of adult female floxed control and conditional knockout (cKO) mice undergoing sham surgery or ovariectomy (OVX). Femoral and tibial bone mineral density (BMD) values were significantly reduced upon OVX in control, but not cKO, females compared to the respective sham-operated mice. Micro-CT analysis showed that OVX significantly increased trabecular number and decreased trabecular spacing in cKO but not control mice. Finally, in control mice serum levels of a marker of bone resorption (pyridinoline crosslinks) and the osteoclast activator RANKL significantly increased upon OVX; however, no such OVX-induced increase was observed in cKO mice. Our results suggest the potential importance of PRKD1 in response to estrogen loss in bone.
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http://dx.doi.org/10.1016/j.mce.2018.03.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6733406PMC
October 2018

Deletion of protein kinase D1 in osteoprogenitor cells results in decreased osteogenesis in vitro and reduced bone mineral density in vivo.

Mol Cell Endocrinol 2018 02 12;461:22-31. Epub 2017 Aug 12.

Institute for Regenerative and Reparative Medicine, Augusta University, 30912, United States; Department of Orthopaedic Surgery, Augusta University, 30912, United States; Department of Neuroscience and Regenerative Medicine, Augusta University, 30912, United States.

Protein kinase D1 (PRKD1) is thought to play a role in a number of cellular functions, including proliferation and differentiation. We hypothesized that PRKD1 in bone marrow-derived mesenchymal stem cells (BMMSC) could modulate osteogenesis. In BMMSCs from floxed PRKD1 mice, PRKD1 ablation with adenovirus-mediated Cre-recombinase expression inhibited BMMSC differentiation in vitro. In 3- and 6-month-old conditional knockout mice (cKO), in which PRKD1 was ablated in osteoprogenitor cells by osterix promoter-driven Cre-recombinase, bone mineral density (BMD) was significantly reduced compared with floxed control littermates. Microcomputed tomography analysis also demonstrated a decrease in trabecular thickness and bone volume fraction in cKO mice at these ages. Dynamic bone histomorphometry suggested a mineralization defect in the cKO mice. However, by 9 months of age, the bone appeared to compensate for the lack of PRKD1, and BMD was not different. Taken together, these results suggest a potentially important role for PRKD1 in bone formation.
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http://dx.doi.org/10.1016/j.mce.2017.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5756499PMC
February 2018

Kynurenine, a Tryptophan Metabolite That Accumulates With Age, Induces Bone Loss.

J Bone Miner Res 2017 Nov 14;32(11):2182-2193. Epub 2017 Aug 14.

Institute for Regenerative and Reparative Medicine, Augusta University, Augusta, GA, USA.

Age-dependent bone loss occurs in humans and in several animal species, including rodents. The underlying causal mechanisms are probably multifactorial, although an age-associated increase in the generation of reactive oxygen species has been frequently implicated. We previously reported that aromatic amino acids function as antioxidants, are anabolic for bone, and that they may potentially play a protective role in an aging environment. We hypothesized that upon oxidation the aromatic amino acids would not only lose their anabolic effects but also potentially become a catabolic byproduct. When measured in vivo in C57BL/6 mice, the tryptophan oxidation product and kynurenine precursor, N-formylkynurenine (NFK), was found to increase with age. We tested the direct effects of feeding kynurenine (kyn) on bone mass and also tested the short-term effects of intraperitoneal kyn injection on bone turnover in CD-1 mice. μCT analyses showed kyn-induced bone loss. Levels of serum markers of osteoclastic activity (pyridinoline [PYD] and RANKL) increased significantly with kyn treatment. In addition, histological and histomorphometric studies showed an increase in osteoclastic activity in the kyn-treated groups in both dietary and injection-based studies. Further, kyn treatment significantly increased bone marrow adiposity, and BMSCs isolated from the kyn-injected mice exhibited decreased mRNA expression of Hdac3 and its cofactor NCoR1 and increased expression of lipid storage genes Cidec and Plin1. A similar pattern of gene expression is observed with aging. In summary, our data show that increasing kyn levels results in accelerated skeletal aging by impairing osteoblastic differentiation and increasing osteoclastic resorption. These data would suggest that kyn could play a role in age-induced bone loss. © 2017 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5685888PMC
November 2017

Mechanisms of acute neurovascular protection with AT1 blockade after stroke: Effect of prestroke hypertension.

PLoS One 2017 22;12(6):e0178867. Epub 2017 Jun 22.

Departments of Biostatistics, Medical College of Georgia, Augusta University, Augusta, Georgia, Unites States of America.

Stroke is a leading cause of adult disability worldwide. Improving stroke outcome requires an orchestrated interplay that involves up regulation of pro-survival pathways and a concomitant suppression of pro-apoptotic mediators. In this investigation, we assessed the involvement of eNOS in the AT1 blocker-mediated protective and pro-recovery effects in animals with hypertension. We also evaluated the effect of acute eNOS inhibition in hypertensive animals. To achieve these goals, spontaneously hypertensive rats (SHR) were implanted with blood pressure transmitters, and randomized to receive either an eNOS inhibitor (L-NIO) or saline one hour before cerebral ischemia induction. After 3 hours of ischemia, animals were further randomized to receive either candesartan or saline at the time of reperfusion and sacrificed either 24 hours or 7 days later. Candesartan induced an early protective effect that was independent of eNOS inhibition (50% improvement in motor function). However, the protective effect of candesartan was associated with about five fold up regulation of BDNF expression and about three fold reduction in ER stress markers, in an eNOS dependent manner. The early benefit of a single dose of candesartan, present at 24 hours after stroke, was diminished at 7 days, perhaps due to a failure to induce an angiogenic response in these hypertensive animals. In conclusion, our findings demonstrate an early prorecovery effect of candesartan at both functional and molecular levels. Candesartan induced prorecovery signaling was mediated through eNOS. This effect was not maintained at 7 days after experimental ischemia.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178867PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5480858PMC
September 2017

MicroRNA-183-5p Increases with Age in Bone-Derived Extracellular Vesicles, Suppresses Bone Marrow Stromal (Stem) Cell Proliferation, and Induces Stem Cell Senescence.

Tissue Eng Part A 2017 11 28;23(21-22):1231-1240. Epub 2017 Apr 28.

Department of Cellular Biology & Anatomy, Medical College of Georgia, Augusta University , Augusta, Georgia .

Microvesicle- and exosome-mediated transport of microRNAs (miRNAs) represents a novel cellular and molecular pathway for cell-cell communication. In this study, we tested the hypothesis that these extracellular vesicles (EVs) and their miRNAs might change with age, contributing to age-related stem cell dysfunction. EVs were isolated from the bone marrow interstitial fluid (supernatant) of young (3-4 months) and aged (24-28 months) mice to determine whether the size, concentration, and miRNA profile of EVs were altered with age in vivo. Results show that EVs isolated from bone marrow are CD63 and CD9 positive, and the concentration and size distribution of bone marrow EVs are similar between the young and aged mice. Bioanalyzer data indicate that EVs from both young and aged mice are highly enriched in miRNAs, and the miRNA profile of bone marrow EVs differs significantly between the young and aged mice. Specifically, the miR-183 cluster (miR-96/-182/-183) is highly expressed in aged EVs. In vitro assays demonstrate that aged EVs are endocytosed by primary bone marrow stromal cells (BMSCs), and these aged EVs inhibit the osteogenic differentiation of young BMSCs. Transfection of BMSCs with miR-183-5p mimic reduces cell proliferation and osteogenic differentiation, increases senescence, and decreases protein levels of the miR-183-5p target heme oxygenase-1 (Hmox1). In vitro assays utilizing HO-induced oxidative stress show that HO treatment of BMSCs increases the abundance of miR-183-5p in BMSC-derived EVs, and Amplex Red assays demonstrate that HO is elevated in the bone marrow microenvironment with age. Together, these data indicate that aging and oxidative stress can significantly alter the miRNA cargo of EVs in the bone marrow microenvironment, which may in turn play a role in stem cell senescence and osteogenic differentiation by reducing Hmox1 activity.
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http://dx.doi.org/10.1089/ten.TEA.2016.0525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5689127PMC
November 2017

Enhanced VEGF signalling mediates cerebral neovascularisation via downregulation of guidance protein ROBO4 in a rat model of diabetes.

Diabetologia 2017 04 23;60(4):740-750. Epub 2017 Jan 23.

Charlie Norwood Veterans Administration Medical Center, Augusta, GA, USA.

Aims/hypothesis: Diabetes promotes cerebral neovascularisation via increased vascular endothelial growth factor (VEGF) angiogenic signalling. Roundabout-4 (ROBO4) protein is an endogenous inhibitor of VEGF signalling that stabilises the vasculature. Yet, how diabetes affects ROBO4 function remains unknown. We hypothesised that increased VEGF signalling in diabetes decreases ROBO4 expression and function via binding of ROBO4 with VEGF-activated β3 integrin and that restoration of ROBO4 expression prevents/repairs cerebral neovascularisation in diabetes.

Methods: ROBO4 protein expression in a rat model of type 2 diabetes (Goto-Kakizaki [GK] rats) was examined by western blotting and immunohistochemistry. ROBO4 was locally overexpressed in the brain and in primary brain microvascular endothelial cells (BMVECs). GK rats were treated with SKLB1002, a selective VEGF receptor-2 (VEGFR-2) antagonist. Cerebrovascular neovascularisation indices were determined using a FITC vascular space-filling model. Immunoprecipitation was used to determine ROBO4-β3 integrin interaction.

Results: ROBO4 expression was significantly decreased in the cerebral vasculature as well as in BMVECs in diabetes (p < 0.05). Silencing Robo4 increased the angiogenic properties of control BMVECs (p < 0.05). In vivo and in vitro overexpression of ROBO4 inhibited VEGF-induced angiogenic signalling and increased vessel maturation. Inhibition of VEGF signalling using SKLB1002 increased ROBO4 expression (p < 0.05) and reduced neovascularisation indices (p < 0.05). Furthermore, SKLB1002 significantly decreased ROBO4-β3 integrin interaction in diabetes (p < 0.05).

Conclusions/interpretation: Our study identifies the restoration of ROBO4 and inhibition of VEGF signalling as treatment strategies for diabetes-induced cerebral neovascularisation.
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http://dx.doi.org/10.1007/s00125-017-4214-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342922PMC
April 2017

Effect of Topical Intranasal Therapy on Epistaxis Frequency in Patients With Hereditary Hemorrhagic Telangiectasia: A Randomized Clinical Trial.

JAMA 2016 09;316(9):943-51

Division of Pulmonary and Critical Care Medicine, Augusta University, Augusta, Georgia.

Importance: Epistaxis is a major factor negatively affecting quality of life in patients with hereditary hemorrhagic telangiectasia (HHT; also known as Osler-Weber-Rendu disease). Optimal treatment for HHT-related epistaxis is uncertain.

Objective: To determine whether topical therapy with any of 3 drugs with differing mechanisms of action is effective in reducing HHT-related epistaxis.

Design, Setting, And Participants: The North American Study of Epistaxis in HHT was a double-blind, placebo-controlled randomized clinical trial performed at 6 HHT centers of excellence. From August 2011 through March 2014, there were 121 adult patients who met the clinical criteria for HHT and had experienced HHT-related epistaxis with an Epistaxis Severity Score of at least 3.0. Follow-up was completed in September 2014.

Interventions: Patients received twice-daily nose sprays for 12 weeks with either bevacizumab 1% (4 mg/d), estriol 0.1% (0.4 mg/d), tranexamic acid 10% (40 mg/d), or placebo (0.9% saline).

Main Outcomes And Measures: The primary outcome was median weekly epistaxis frequency during weeks 5 through 12. Secondary outcomes included median duration of epistaxis during weeks 5 through 12, Epistaxis Severity Score, level of hemoglobin, level of ferritin, need for transfusion, emergency department visits, and treatment failure.

Results: Among the 121 patients who were randomized (mean age, 52.8 years [SD, 12.9 years]; 44% women with a median of 7.0 weekly episodes of epistaxis [interquartile range {IQR}, 3.0-14.0]), 106 patients completed the study duration for the primary outcome measure (43 were women [41%]). Drug therapy did not significantly reduce epistaxis frequency (P = .97). After 12 weeks of treatment, the median weekly number of bleeding episodes was 7.0 (IQR, 4.5-10.5) for patients in the bevacizumab group, 8.0 (IQR, 4.0-12.0) for the estriol group, 7.5 (IQR, 3.0-11.0) for the tranexamic acid group, and 8.0 (IQR, 3.0-14.0) for the placebo group. No drug treatment was significantly different from placebo for epistaxis duration. All groups had a significant improvement in Epistaxis Severity Score at weeks 12 and 24. There were no significant differences between groups for hemoglobin level, ferritin level, treatment failure, need for transfusion, or emergency department visits.

Conclusions And Relevance: Among patients with HHT, there were no significant between-group differences in the use of topical intranasal treatment with bevacizumab vs estriol vs tranexamic acid vs placebo and epistaxis frequency.

Trial Registration: clinicaltrials.gov Identifier: NCT01408030.
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http://dx.doi.org/10.1001/jama.2016.11724DOI Listing
September 2016

Nox4 contributes to the hypoxia-mediated regulation of actin cytoskeleton in cerebrovascular smooth muscle.

Life Sci 2016 Oct 21;163:46-54. Epub 2016 Aug 21.

Charlie Norwood VA Medical Center, Augusta University, United States; Departments of Physiology, Augusta University, United States; Program in Clinical and Experimental Therapeutics, University of Georgia College of Pharmacy, Augusta, GA, United States. Electronic address:

Unlabelled: Ischemia/reperfusion and the resulting oxidative/nitrative stress impair cerebral myogenic tone via actin depolymerization. While it is known that NADPH oxidase (Nox) family is a major source of vascular oxidative stress; the extent and mechanisms by which Nox activation contributes to actin depolymerization, and equally important, the relative role of Nox isoforms in this response is not clear.

Aim: To determine the role of Nox4 in hypoxia-mediated actin depolymerization and myogenic-tone impairment in cerebral vascular smooth muscle.

Main Methods: Control and Nox4 deficient (siRNA knock-down) human brain vascular smooth muscle cells (HBVSMC) were exposed to 30-min hypoxia/45-min reoxygenation. Nox2, Nox4, inducible and neuronal nitric oxide synthase (iNOS and nNOS) and nitrotyrosine levels as well as F:G actin were determined. Myogenic-tone was measured using pressurized arteriography in middle cerebral artery isolated from rats subjected to sham, 30-min ischemia/45-min reperfusion or ex-vivo oxygen glucose deprivation in the presence and absence of Nox inhibitors.

Results: Nox4 and iNOS expression were significantly upregulated following hypoxia or ischemia/reperfusion. Hypoxia augmented nitrotyrosine levels while reducing F actin. These effects were nullified by inhibiting nitration with epicatechin or pharmacological or molecular inhibition of Nox4. Ischemia/reperfusion impaired myogenic-tone, which was restored by the selective inhibition of Nox4.

Conclusion: Nox4 activation in VSMCs contributes to actin depolymerization after hypoxia, which could be the underlying mechanism for myogenic-tone impairment following ischemia/reperfusion.
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http://dx.doi.org/10.1016/j.lfs.2016.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5028312PMC
October 2016

Linagliptin treatment improves cerebrovascular function and remodeling and restores reduced cerebral perfusion in Type 2 diabetes.

Am J Physiol Regul Integr Comp Physiol 2016 09 29;311(3):R466-77. Epub 2016 Jun 29.

Charlie Norwood Veterans Administration Medical Center, Augusta, Georgia; Department of Physiology, Medical College of Georgia, Augusta, Georgia; and

The antihyperglycemic agent linagliptin, a dipeptidyl peptidase-4 (DPP-IV) inhibitor, has been shown to reduce inflammation and improve endothelial cell function. In this study, we hypothesized that DPP-IV inhibition with linagliptin would improve impaired cerebral perfusion in diabetic rats, as well as improve insulin-induced cerebrovascular relaxation and reverse pathological cerebrovascular remodeling. We further postulated that these changes would lead to a subsequent improvement of cognitive function. Male Type-2 diabetic and nondiabetic Goto-Kakizaki rats were treated with linagliptin for 4 wk, and blood glucose and DPP-IV plasma levels were assessed. Cerebral perfusion was assessed after treatment using laser-Doppler imaging, and dose response to insulin (10(-13) M-10(-6) M) in middle cerebral arteries was tested on a pressurized arteriograph. The impact of DPP-IV inhibition on diabetic cerebrovascular remodeling was assessed over a physiologically relevant pressure range, and changes in short-term hippocampus-dependent learning were observed using a novel object recognition test. Linagliptin lowered DPP-IV activity but did not change blood glucose or insulin levels in diabetes. Insulin-mediated vascular relaxation and cerebral perfusion were improved in the diabetic rats with linagliptin treatment. Indices of diabetic vascular remodeling, such as increased cross-sectional area, media thickness, and wall-to-lumen ratio, were also ameliorated; however, improvements in short-term hippocampal-dependent learning were not observed. The present study provides evidence that linagliptin treatment improves cerebrovascular dysfunction and remodeling in a Type 2 model of diabetes independent of glycemic control. This has important implications in diabetic patients who are predisposed to the development of cerebrovascular complications, such as stroke and cognitive impairment.
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http://dx.doi.org/10.1152/ajpregu.00057.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5142225PMC
September 2016

VLDL-activated cell signaling pathways that stimulate adrenal cell aldosterone production.

Mol Cell Endocrinol 2016 09 21;433:138-46. Epub 2016 May 21.

Charlie Norwood VA Medical Center, One Freedom Way, Augusta, GA, 30904, United States; Department of Physiology, Medical College of Georgia at Augusta University, 1120 15th Street, Augusta, GA, 30912, United States. Electronic address:

Aldosterone plays an important role in regulating ion and fluid homeostasis and thus blood pressure, and hyperaldosteronism results in hypertension. Hypertension is also observed with obesity, which is associated with additional health risks, including cardiovascular disease. Obese individuals have high serum levels of very low-density lipoprotein (VLDL), which has been shown to stimulate aldosterone production; however, the mechanisms underlying VLDL-induced aldosterone production are still unclear. Here we demonstrate in human adrenocortical carcinoma (HAC15) cells that submaximal concentrations of angiotensin II and VLDL stimulate aldosterone production in an additive fashion, suggesting the possibility of common mechanisms of action. We show using inhibitors that VLDL-induced aldosterone production is mediated by the PLC/IP3/PKC signaling pathway. Our results suggest that PKC is upstream of the extracellular signal-regulated kinase (ERK) activation previously observed with VLDL. An understanding of the mechanisms mediating VLDL-induced aldosterone production may provide insights into therapies to treat obesity-associated hypertension.
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http://dx.doi.org/10.1016/j.mce.2016.05.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4955520PMC
September 2016

Matrix Metalloprotease 3 Exacerbates Hemorrhagic Transformation and Worsens Functional Outcomes in Hyperglycemic Stroke.

Stroke 2016 Mar 2;47(3):843-51. Epub 2016 Feb 2.

From the Charlie Norwood VA Medical Center (S.H., S.C.F., A.E.), Departments of Physiology (S.H., M.A., S.E.-S., A.E.), Biostatistics (M.H.J.), and Neurology (S.C.F.), Medical College of Georgia, Augusta University; and Program in Clinical and Experimental Therapeutics, University of Georgia College of Pharmacy, Augusta (S.H., S.C.F., A.E.).

Background And Purpose: Acute hyperglycemia worsens the clinical outcomes and exacerbates cerebral hemorrhage after stroke. The mediators of hemorrhagic transformation (HT) in hyperglycemic stroke are not fully understood. Matrix metalloproteinase 3 (MMP3) plays a critical role in the tissue-type plasminogen activator-induced HT. However, the role of MMP3 in exacerbating the HT and worsening the functional outcomes in hyperglycemic stroke remains unknown.

Methods: Control/normoglycemic and hyperglycemic (blood glucose, 140-200 mg/dL) male Wistar rats were subjected to middle cerebral artery occlusion for 90 minutes and either 24 hours or 7 days reperfusion. MMP3 was inhibited pharmacologically (UK 356618, 15 mg/kg IV at reperfusion) or knocked down in the brain by shRNA lentiviral particles (injected intracerebroventricular). Neurovascular injury was assessed at 24 hours, and functional outcomes were assessed at 24 hours, day 3, and day 7. MMP3 activity was measured in brain homogenate and cerebral macrovessels. Localization of MMP3 within the neurovascular unit after hyperglycemic stroke was demonstrated by immunohistochemistry.

Results: Hyperglycemia significantly increased MMP3 activity in the brain after stroke, and this was associated with exacerbated HT and worsened functional outcomes. MMP3 inhibition significantly reduced HT and improved functional outcomes.

Conclusions: MMP3 plays a critical role in mediating cerebrovascular injury in hyperglycemic stroke. Our findings point out MMP3 as a potential therapeutic target in hyperglycemic stroke.
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http://dx.doi.org/10.1161/STROKEAHA.115.011258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4766051PMC
March 2016

The aromatic amino acid tryptophan stimulates skeletal muscle IGF1/p70s6k/mTor signaling in vivo and the expression of myogenic genes in vitro.

Nutrition 2015 Jul-Aug;31(7-8):1018-24. Epub 2015 Mar 17.

Georgia Regents University (Formerly Georgia Health Sciences University), Augusta, GA, USA. Electronic address:

Objectives: Nutrition plays a key role in the maintenance of muscle and bone mass, and dietary protein deficiency has in particular been associated with catabolism of both muscle and bone tissue. One mechanism thought to link protein deficiency with loss of muscle mass is deficiency in specific amino acids that play a role in muscle metabolism. The aim of this study was to test the hypothesis that the essential amino acid tryptophan, and its metabolite kynurenine, might directly affect muscle metabolism in the setting of protein deficiency.

Methods: Adult mice (12 mo) were fed a normal diet (18% protein), as well as diets with low protein (8%) supplemented with increasing concentrations (50, 100, and 200 uM) of kynurenine (Kyn) or with tryptophan (Trp; 1.5 mM) for 8 weeks. Myoprogenitor cells were also treated with Trp and Kyn in vitro to determine their effects on cell proliferation and expression of myogenic differentiation markers.

Results: All mice on the low-protein diets weighed less than the group fed normal protein (18%). Lean mass measured by dual-energy X-ray absorptiometry was lowest in mice on the high Kyn diet, whereas percent lean mass was highest in mice receiving Trp supplementation and percent body fat was lowest in mice receiving Trp. Enzyme-linked immunosorbent assays showed significant increases in skeletal muscle insulin-like growth factor-1, leptin, and the myostatin antagonist follistatin with Trp supplementation. mRNA microarray and gene pathway analysis performed on muscle samples demonstrate that mTor/eif4/p70s6k pathway molecules are significantly up-regulated in muscles from mice on Kyn and Trp supplementation. In vitro, neither amino acid affected proliferation of myoprogenitors, but Trp increased the expression of the myogenic markers MyoD, myogenin, and myosin heavy chain.

Conclusion: These findings suggest that dietary amino acids can directly affect molecular signaling in skeletal muscle, further indicating that dietary manipulation with specific amino acids could potentially attenuate muscle loss with dietary protein deficiency.
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http://dx.doi.org/10.1016/j.nut.2015.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4465076PMC
May 2016

Clinical and economic burden of community-acquired pneumonia in the Veterans Health Administration, 2011: a retrospective cohort study.

Infection 2015 Dec 17;43(6):671-80. Epub 2015 May 17.

Charlie Norwood VA Medical Center, Augusta, GA, USA.

Purpose: The burden of community-acquired pneumonia (CAP) is not well described in the US Veterans Health Administration (VHA).

Methods: CAP was defined as having a pneumonia diagnosis with evidence of chest X-ray, and no evidence of prior (90 days) hospitalization/long-term care. We calculated incidence rates of adult CAP occurring in inpatient or outpatient VHA settings in 2011. We also estimated the proportion of VHA CAP patients who were hospitalized, were readmitted within 30 days of hospital discharge, and died (any cause) in the year following diagnosis. Incremental costs during the 90 days following a CAP diagnosis were estimated from the perspective of the VHA.

Results: In 2011, 34,101 Veterans developed CAP (35,380 episodes) over 7,739,757 VHA person-years. Median age of CAP patients was 65 years (95 % male). CAP incidence rates were higher for those aged ≥50 years. A majority of Veterans aged 50-64 (53 %) and ≥65 (66 %) years had ≥1 chronic medical (moderate risk) or immunocompromising (high risk) condition. Compared to those at low-risk (healthy), moderate- and high-risk Veterans were >3 and >6 times more likely to develop CAP, respectively. The percentage of CAP patients who were hospitalized was 45 %, ranging from 12 % (age 18-49, low risk) to 57 % (age ≥65, high risk). One-year all-cause mortality rates ranged from 1 % (age 18-49, low risk) to 36 % (age ≥65, high risk). Annual VHA medical expenditure related to CAP was estimated to be $750 million (M) ($415M for those aged ≥65 years).

Conclusion: A focus on CAP prevention among older Veterans and those with comorbid or immunocompromising conditions is important.
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http://dx.doi.org/10.1007/s15010-015-0789-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4656694PMC
December 2015

Comparative Analysis of Different Methods of Ischemia/Reperfusion in Hyperglycemic Stroke Outcomes: Interaction with tPA.

Transl Stroke Res 2015 Jun 17;6(3):171-80. Epub 2015 Feb 17.

Charlie Norwood Veterans Administration Medical Center, Augusta, GA, USA.

Acute hyperglycemia (HG) exacerbates reperfusion injury and aggravates tissue plasminogen activator (tPA)-induced hemorrhagic transformation (HT). Previous experimental hyperglycemic stroke studies employed very high blood glucose levels and exclusively used suture occlusion model to induce ischemia. Only few studies evaluated HG in embolic stroke and mostly involving the use of 10-fold higher dose of tPA than that is used in patients. However, the interaction between acute HG and low (human) dose tPA in different experimental models of stroke has never been reported. We first tested the impact of the severity of acute HG on stroke outcome. Building upon our findings, we then compared the impact of mild acute HG on neurovascular injury in rats subjected to suture or thromboembolic occlusion with and without low dose tPA. We assessed cerebral blood flow, neurobehavioral outcomes, infarction, hemorrhage, and edema. tPA did not change the infarct size in either control or hyperglycemic animals when compared to no tPA groups. HG increased HT and worsened functional outcomes in both suture and embolic occlusion models. The combination of HG and tPA exacerbated the vascular injury and worsened the neurological deficits more than each individual treatment in both models. Our findings show that the interaction between HG and even low dose tPA has detrimental effects on the cerebrovasculature and functional outcomes independent of the method of reperfusion.
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http://dx.doi.org/10.1007/s12975-015-0391-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4425641PMC
June 2015

SOD1 overexpression prevents acute hyperglycemia-induced cerebral myogenic dysfunction: relevance to contralateral hemisphere and stroke outcomes.

Am J Physiol Heart Circ Physiol 2015 Mar 31;308(5):H456-66. Epub 2014 Dec 31.

Charlie Norwood Veterans Affairs Medical Center, Augusta, Georgia; Department of Physiology, Georgia Regents University, Augusta, Georgia; Program in Clinical and Experimental Therapeutics, University of Georgia College of Pharmacy, Augusta, Georgia

Admission hyperglycemia (HG) amplifies vascular injury and neurological deficits in acute ischemic stroke, but the mechanisms remain controversial. We recently reported that ischemia-reperfusion (I/R) injury impairs the myogenic response in both hemispheres via increased nitration. However, whether HG amplifies contralateral myogenic dysfunction and whether loss of tone in the contralateral hemisphere contributes to stroke outcomes remain to be determined. Our hypothesis was that contralateral myogenic dysfunction worsens stroke outcomes after acute hyperglycemic stroke in an oxidative stress-dependent manner. Male wild-type or SOD1 transgenic rats were injected with saline or 40% glucose solution 10 min before surgery and then subjected to 30 min of ischemia/45 min or 24 h of reperfusion. In another set of animals (n = 5), SOD1 was overexpressed only in the contralateral hemisphere by stereotaxic adenovirus injection 2-3 wk before I/R. Myogenic tone and neurovascular outcomes were determined. HG exacerbated myogenic dysfunction in contralateral side only, which was associated with infarct size expansion, increased edema, and more pronounced neurological deficit. Global and selective SOD1 overexpression restored myogenic reactivity in ipsilateral and contralateral sides, respectively, and enhanced neurovascular outcomes. In conclusion, our results show that SOD1 overexpression nullified the detrimental effects of HG on myogenic tone and stroke outcomes and that the contralateral hemisphere may be a novel target for the management of acute hyperglycemic stroke.
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http://dx.doi.org/10.1152/ajpheart.00321.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4346767PMC
March 2015

Metformin treatment in the period after stroke prevents nitrative stress and restores angiogenic signaling in the brain in diabetes.

Diabetes 2015 May 18;64(5):1804-17. Epub 2014 Dec 18.

Charlie Norwood Veterans Administration Medical Center, Augusta, GA Department of Physiology, Georgia Regents University, Augusta, GA Program in Clinical and Experimental Therapeutics, University of Georgia College of Pharmacy, Augusta, GA

Diabetes impedes vascular repair and causes vasoregression in the brain after stroke, but mechanisms underlying this response are still unclear. We hypothesized that excess peroxynitrite formation in diabetic ischemia/reperfusion (I/R) injury inactivates the p85 subunit of phosphoinositide 3-kinase (PI3K) by nitration and diverts the PI3K-Akt survival signal to the p38-mitogen-activated protein kinase apoptosis pathway. Nitrotyrosine (NY), Akt and p38 activity, p85 nitration, and caspase-3 cleavage were measured in brains from control, diabetic (GK), or metformin-treated GK rats subjected to sham or stroke surgery and in brain microvascular endothelial cells (BMVECs) from Wistar and GK rats subjected to hypoxia/reoxygenation injury. GK rat brains showed increased NY, caspase-3 cleavage, and p38 activation and decreased Akt activation. Metformin attenuated stroke-induced nitrative signaling in GK rats. GK rat BMVECs showed increased basal nitrative stress compared with controls. A second hit by hypoxia/reoxygenation injury dramatically increased the nitration of p85 and activation of p38 but decreased Akt. These effects were associated with impairment of angiogenic response and were restored by treatment with the peroxynitrite scavenger 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chloride or the nitration inhibitor epicatechin. Our results provide evidence that I/R-induced peroxynitrite inhibits survival, induces apoptosis, and promotes peroxynitrite as a novel therapeutic target for the improvement of reparative angiogenesis after stroke in diabetes.
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http://dx.doi.org/10.2337/db14-1423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407857PMC
May 2015

Mesenchymal stem cell expression of stromal cell-derived factor-1β augments bone formation in a model of local regenerative therapy.

J Orthop Res 2015 Feb 28;33(2):174-84. Epub 2014 Oct 28.

Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio.

Bone has the potential for spontaneous healing. However, this process often fails in patients with co-morbidities requiring clinical intervention. Numerous studies have revealed that bone marrow-derived mesenchymal stem/stromal cells (BMSCs) hold great potential for regenerative therapies. Common problems include poor cell engraftment, which can be addressed by irradiation prior to transplantation. Increasing evidence suggests that stromal cell-derived factor-1 (SDF-1) is involved in bone formation. However, osteogenic contributions of the beta splice variant of SDF-1 (SDF-1β), which is highly expressed in bone, remain unclear. Using the tetracycline (Tet)-regulatory system we have shown that SDF-1β enhances BMSC osteogenic differentiation in vitro. Here we test the hypothesis that SDF-1β augments bone formation in vivo in a model of local BMSC transplantation following irradiation. We found that SDF-1β, expressed at high levels in Tet-Off-SDF-1β BMSCs, augments the cell-mediated therapeutic effects resulting in enhanced bone formation, as evidenced by ex vivo μCT and bone histomorphometry. The data demonstrate the specific contribution of SDF-1β to BMSC-mediated bone formation, and validate the feasibility of the Tet-Off technology to regulate SDF-1β expression in vivo. In conclusion, SDF-1β provides potent synergistic effects supporting BMSC-mediated bone formation and appears a suitable candidate for optimization of bone augmentation in translational protocols.
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http://dx.doi.org/10.1002/jor.22749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706461PMC
February 2015
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