Mamatha Damodarasamy

Lipopolysaccharide-induced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit

BackgroundDisruption of the blood-brain barrier (BBB) occurs in many diseases and is often mediated by inflammatory and neuroimmune mechanisms. Inflammation is well established as a cause of BBB disruption, but many mechanistic questions remain.MethodsWe used lipopolysaccharide (LPS) to induce inflammation and BBB disruption in mice. BBB disruption was measured using 14C-sucrose and radioactively labeled albumin. Brain cytokine responses were measured using multiplex technology and dependence on cyclooxygenase (COX) and oxidative stress determined by treatments with indomethacin and N-acetylcysteine. Astrocyte and microglia/macrophage responses were measured using brain immunohistochemistry. In vitro studies used Transwell cultures of primary brain endothelial cells co- or tri-cultured with astrocytes and pericytes to measure effects of LPS on transendothelial electrical resistance (TEER), cellular distribution of tight junction proteins, and permeability to 14C-sucrose and radioactive albumin.ResultsIn comparison to LPS-induced weight loss, the BBB was relatively resistant to LPS-induced disruption. Disruption occurred only with the highest dose of LPS and was most evident in the frontal cortex, thalamus, pons-medulla, and cerebellum with no disruption in the hypothalamus. The in vitro and in vivo patterns of LPS-induced disruption as measured with 14C-sucrose, radioactive albumin, and TEER suggested involvement of both paracellular and transcytotic pathways. Disruption as measured with albumin and 14C-sucrose, but not TEER, was blocked by indomethacin. N-acetylcysteine did not affect disruption. In vivo, the measures of neuroinflammation induced by LPS were mainly not reversed by indomethacin. In vitro, the effects on LPS and indomethacin were not altered when brain endothelial cells (BECs) were cultured with astrocytes or pericytes.ConclusionsThe BBB is relatively resistant to LPS-induced disruption with some brain regions more vulnerable than others. LPS-induced disruption appears is to be dependent on COX but not on oxidative stress. Based on in vivo and in vitro measures of neuroinflammation, it appears that astrocytes, microglia/macrophages, and pericytes play little role in the LPS-mediated disruption of the BBB.

CCL5 secreted by senescent aged fibroblasts induces proliferation of prostate epithelial cells and expression of genes that modulate angiogenesis

There is increased interest in the effects of secretory products from aged cells on promoting both benign and malignant cell growth. We identified a human fibroblast line, AG04382, from an aged donor that naturally demonstrated senescence‐associated features and whose conditioned media significantly induced proliferation of benign prostatic hyperplasia (BPH1) cells. Candidate cytokines mediating this effect were identified with protein arrays and validated by ELISA. We found that the AG04382 fibroblast line secreted high levels of CXCL5, CCL5, and CCL2, but relative to the other lines, its conditioned media was unique in its increased expression of CCL5. Blocking studies using specific antibodies against CXCL5, CCL5, and CCL2 in the conditioned media of AG04382 showed that only CCL5 contributed significantly to BPH1 proliferation. Stimulation of BPH1 cells with rhuCCL5 resulted in increased proliferation and migration, as well as significant changes in the expression of genes that influence angiogenesis. These data suggest that CCL5 is a candidate chemokine secreted by aged cells that promotes prostate growth and regulates angiogenesis. J. Cell. Physiol. 220: 376–381, 2009.

Collagen extracts derived from young and aged mice demonstrate different structural properties and cellular effects in three-dimensional gels.

Three-dimensional (3D) type I collagen gels are increasingly utilized to simulate extracellular matrix (ECM) in vivo, but little is known about the effects of age on this model. Collagen was extracted from young (4-6 months) and aged (20-24 months) mice tails and compared. The collagens appeared similar by electrophoresis. However, relative to young, aged collagen formed fibrils slower and generated 3D gels with smaller diameter, less dense fibrils (75 vs 34 nm diameter and 8 vs 3.5% area, for young and aged respectively, p < 0.02). Correspondingly, aged collagen gels were more malleable and contractible (5% vs 19% compression, p < .02, and 73% vs 15.5% area, p < .01, for young and aged, respectively). Fibroblasts cultured within young and aged collagen gels had differential expression of a limited number of genes and proteins corresponding to specific integrins and matrix components. In summary, collagen extracted from young and aged mice is an effective means to examine the influence of aging on functional properties of ECM that are relevant in vivo.

Hyaluronan enhances wound repair and increases collagen III in aged dermal wounds

Age‐related changes in the extracellular matrix contribute to delayed wound repair in aging. Hyaluronan, a linear nonsulfated glycosaminoglycan, promotes synthesis and assembly of key extracellular matrix components, such as the interstitial collagens, during wound healing. The biological effects of hyaluronan are mediated, in part, by hyaluronan size. We have previously determined that dermal wounds in aged mice, relative to young mice, have deficits in the generation of lower molecular weight hyaluronan (defined as <300 kDa). Here, we tested the effect of exogenous hyaluronan of 2, 250, or 1,000 kDa sizes on full‐thickness excisional wounds in aged mice. Only wounds treated with 250 kDa hyaluronan (HA250) were significantly improved over wounds that received carrier (water) alone. Treatment with HA250 was associated with increased expression of transcripts for the hyaluronan receptors CD44 and RHAMM, as well as collagens III and I. Analyses of dermal protein content by mass spectrometry and Western blotting confirmed significantly increased expression of collagen III in wounds treated with HA250 relative to control wounds. In summary, we find that HA250 improves wound repair and increases the synthesis of collagen III in aged dermal wounds.

Association of Radiologic Indicators of Frailty With 1-Year Mortality in Older Trauma Patients: Opportunistic Screening for Sarcopenia and Osteopenia

Importance Assessment of physical frailty in older trauma patients admitted to the intensive care unit is often not feasible using traditional frailty assessment instruments. The use of opportunistic computed tomography (CT) scans to assess sarcopenia and osteopenia as indicators of underlying frailty may provide complementary prognostic information on long-term outcomes. Objective To determine whether sarcopenia and/or osteopenia are associated with 1-year mortality in an older trauma patient population. Design, Setting, and Participants A retrospective cohort constructed from a state trauma registry was linked to the statewide death registry and Comprehensive Hospital Abstract Reporting System for readmission data analyses. Admission abdominopelvic CT scans from patients 65 years and older admitted to the intensive care unit of a single level I trauma center between January 2011 and May 2014 were analyzed to identify patients with sarcopenia and/or osteopenia. Patients with a head Injury Severity Score of 3 or greater, an out-of-state address, or inadequate CT imaging or who died within 24 hours of admission were excluded. Exposures Sarcopenia and/or osteopenia, assessed via total cross-sectional muscle area and bone density at the L3 vertebral level, compared with a group with no sarcopenia or osteopenia. Main Outcomes and Measures One-year all-cause mortality. Secondary outcomes included 30-day all-cause mortality, 30-day readmission, hospital length of stay, hospital cost, and discharge disposition. Results Of the 450 patients included in the study, 269 (59.8%) were male and 394 (87.6%) were white. The cohort was split into 4 groups: 74 were retrospectively diagnosed with both sarcopenia and osteopenia, 167 with sarcopenia only, 48 with osteopenia only, and 161 with no radiologic indicators. Among the 408 who survived to discharge, sarcopenia and osteopenia were associated with higher risks of 1-year mortality alone and in combination. After adjustment, the hazard ratio was 9.4 (95% CI, 1.2-75.4; P = .03) for sarcopenia and osteopenia, 10.3 (95% CI, 1.3-78.8; P = .03) for sarcopenia, and 11.9 (95% CI, 1.3-107.4; P = .03) for osteopenia. Conclusions and Relevance More than half of older trauma patients in this study had sarcopenia, osteopenia, or both. Each factor was independently associated with increased 1-year mortality. Given the prevalent use of abdominopelvic CT in trauma centers, opportunistic screening for radiologic indicators of frailty provides an additional tool for early identification of older trauma patients at high risk for poor outcomes, with the potential for targeted interventions.

Decreased proliferative capacity of aged dermal fibroblasts in a three dimensional matrix is associated with reduced IGF1R expression and activation

Skin aging results in increased susceptibility to injury and impaired wound healing. Proliferation of fibroblasts is reduced in aged dermis, which contributes to delays in wound closure. Age-associated differences are regulated, in part, by local or systemic factors such as the IGF-1/IGF1R system. The aim of this study was to determine if expression and activation of IGF1R in aged human dermal fibroblasts, when compared to young fibroblasts, is associated with altered proliferative capacity in a 3D collagen matrix that better simulates the dermal extracellular matrix in vivo. The proliferation of young and aged human dermal fibroblasts in 3D collagen and its association with baseline levels of IGF1R expression were measured. The effect of stimulation and inhibition of Erk phosphorylation on the proliferative capacity of fibroblasts in a 3D collagen matrix was defined. Our results show that proliferation and Erk phosphorylation is reduced in aged dermal fibroblasts relative to young fibroblasts. Activation of Erk phosphorylation in aged fibroblasts is associated with a significant increase in fibroblast proliferation in 3D collagen.

Angiogenesis In Vitro Utilizing Murine Vascular Explants in Miniaturized 3-Dimensional Collagen Gels.

Models of angiogenesis in vitro are used to study blood vessel morphogenesis and the effects of compounds that influence vascular growth. Herein, we describe techniques to induce angiogenesis-like sprouting from explants of mouse aortae and microvessels cultured in 3-dimensional gels of native type I collagen. The gels are supported by rings of nylon mesh that are sized to fit in 96-well culture plates. This mechanically-supported, miniaturized, 3-dimensional culture system requires only small quantities of cells and reagents and facilitates handling, staining, and imaging by conventional and confocal microscopy.

Lidocaine Impairs Proliferative and Biosynthetic Functions of Aged Human Dermal Fibroblasts

BACKGROUND: The aged are at increased risk of postoperative wound healing complications. Because local anesthetics are infiltrated commonly into the dermis of surgical wounds, we sought to determine whether local anesthetics adversely affect proliferative and biosynthetic functions of dermal fibroblasts. We also evaluated the effect of local anesthetics on insulin-like growth factor-1 (IGF-1) and transforming growth factor-&bgr;1 (TGF-&bgr;1), growth factors that are important regulators of wound healing. METHODS: Human dermal fibroblasts (HFB) from aged and young donors were exposed to local anesthetic agents at clinically relevant concentrations. We screened the effects of lidocaine, bupivacaine, mepivacaine, and ropivacaine on proliferation of HFB. Lidocaine was most detrimental to proliferation in HFB. We then evaluated the effect of lidocaine on expression and function of the growth factors, IGF-1 and TGF-&bgr;1. Lastly, concurrent exposure to lidocaine and IGF-1 or TGF-&bgr;1 was evaluated for their effects on proliferation and expression of dermal collagens, respectively. RESULTS: Lidocaine and mepivacaine inhibited proliferation in aged HFB (for lidocaine 88% of control, 95% confidence interval [CI], 80%–98%, P = .009 and for mepivacaine 90% of control, 95% CI, 81%–99%, P = .032) but not in young HFB. Ropivacaine and bupivacaine did not inhibit proliferation. Because of the clinical utility of lidocaine relative to mepivacaine, we focused on lidocaine. Lidocaine decreased proliferation in aged HFB, which was abrogated by IGF-1. Lidocaine inhibited transcripts for IGF-1 and insulin-like growth factor-1 receptor (IGF1R) in fibroblasts from aged donors (IGF-1, log2 fold-change −1.25 [42% of control, 95% CI, 19%–92%, P = .035] and IGF1R, log2 fold-change −1.00 [50% of control, 95% CI, 31%–81%, P = .014]). In contrast, lidocaine did not affect the expression of IGF-1 or IGF1R transcripts in the young HFB. Transcripts for collagen III were decreased after lidocaine exposure in aged and young HFB (log2 fold-change −1.28 [41% of control, 95% CI, 20%–83%, P = .022] in aged HFB and log2 fold-change −1.60 [33% of control, 95% CI, 15%–73%, P = .019] in young HFB). Transcripts for collagen I were decreased in aged HFB (log2 fold-change −1.82 [28% of control, 95% CI, 14%–58%, P = .006]) but not in the young HFB. Similar to the transcripts, lidocaine also inhibited the protein expression of collagen III in young and aged HFB (log2 fold-change −1.79 [29% of control, 95% CI, 18%–47%, P = .003] in young HFB and log2 fold-change −1.76 [30% of control, 95% CI, 9%–93%, P = .043] in aged HFB). The effect of lidocaine on the expression of collagen III protein was obviated by TGF-&bgr;1 in both young and aged HFB. CONCLUSIONS: Our results show that lidocaine inhibits processes relevant to dermal repair in aged HFB. The detrimental responses to lidocaine are due, in part, to interactions with IGF-1 and TGF-&bgr;1.

Hyaluronan in aged collagen matrix increases prostate epithelial cell proliferation.

The extracellular matrix (ECM) of the prostate, which is comprised primarily of collagen, becomes increasingly disorganized with age, a property that may influence the development of hyperplasia and cancer. Collageous ECM extracted from the tails of aged mice exhibits many characteristics of collagen in aged tissues, including the prostate. When polymerized into a 3-dimensional (3D) gel, these collagen extracts can serve as models for the study of specific cell-ECM interactions. In the present study, we examined the behaviors of human prostatic epithelial cell lines representing normal prostate epithelial cells (PEC), benign prostatic hyperplasia (BPH-1), and adenocarcinoma (LNCaP) cultured in contact with 3D gels made from collagen extracts of young and aged mice. We found that proliferation of PEC, BPH-1, and LNCaP cells were all increased by culture on aged collagen gels relative to young collagen gels. In examining age-associated differences in the composition of the collagen extracts, we found that aged and young collagen had a similar amount of several collagen-associated ECM components, but aged collagen had a much greater content of the glycosaminoglycan hyaluronan (HA) than young collagen. The addition of HA (of similar size and concentration to that found in aged collagen extracts) to cells placed in young collagen elicited significantly increased proliferation in BPH-1 cells, but not in PEC or LNCaP cells, relative to controls not exposed to HA. Of note, histochemical analyses of human prostatic tissues showed significantly higher expression of HA in BPH and prostate cancer stroma relative to stroma of normal prostate. Collectively, these results suggest that changes in ECM involving increased levels of HA contribute to the growth of prostatic epithelium with aging.

The Effects of Normal Aging on Regional Accumulation of Hyaluronan and Chondroitin Sulfate Proteoglycans in the Mouse Brain

The brain changes in volume and composition with normal aging. Cellular components of the brain are supported by an extracellular matrix (ECM) comprised largely of hyaluronan (HA) and HA-associated members of the lectican family of chondroitin sulfate proteoglycans (CSPGs). We examined regional differences in microvascular density, neuronal and glial markers, and accumulation of HA and CSPGs in mouse brains during normal aging. The cortex, hippocampus, dentate gyrus, and cerebellum of young (4 months), middle-aged (14 months), and aged (24–26 months) brains were analyzed. Microvascular density decreased in cerebral cortex and cerebellum with age. There were no detectable differences in neuronal density. There was an increase in astrocytes in the hippocampus with aging. HA accumulation was higher in aged brain relative to young brain in the cerebral cortex and cerebellum, but not in other regions examined. In contrast, CSPGs did not change with aging in any of the brain regions examined. HA and CSPGs colocalized with a subset of neuronal cell bodies and astrocytes, and at the microvasculature. Differences in accumulation of ECM in the aging brain, in the setting of decreased microvascular density and/or increased glial activation, might contribute to age-related regional differences in vulnerability to injury and ischemia.