ABSTRACT
Ischemic stroke remains a leading cause of mortality and long-term disability worldwide, necessitating efforts to identify biomarkers for diagnosis, prognosis, and treatment monitoring. The present study aimed to identify novel plasma biomarkers of neurodegeneration and inflammation in a mouse model of stroke induced by distal middle cerebral artery (MCA) occlusion. Using targeted lipidomic and global untargeted metabolomic profiling of plasma collected from aged male mice 24 hours after stroke and weekly thereafter for 7 weeks, we discovered distinct acute and chronic signatures. In the acute phase, we observed elevations in myelin-associated lipids, including sphingomyelin (SM) and hexosylceramide (HCER) lipid species, indicating brain lipid catabolism. In the chronic phase, we identified 12-hydroxyeicosatetraenoic acid (12-HETE) as a putative biomarker of prolonged inflammation, consistent with our previous observation of a biphasic pro-inflammatory response to ischemia in the mouse brain. These results provide insight into the metabolic alterations detectable in the plasma after stroke and highlight the potential of myelin degradation products and arachidonic acid derivatives as biomarkers of neurodegeneration and inflammation, respectively. These discoveries lay the groundwork for further validation in human studies and may improve stroke management strategies.
ABSTRACT
Secondary spinal cord injury (SCI) is characterized by increased cytokines and chemokines at the site of injury that have been associated with the development of neuropathic pain. Nearly 80% of SCI patients report suffering from chronic pain, which is poorly managed with available analgesics. While treatment with the FDA-approved ß2-adrenergic receptor agonist, formoterol, improves various aspects of recovery post-SCI in vivo, its effects on cytokines, chemokines and neuropathic pain remain unknown. Female mice were subjected to moderate (60 kdyn) or severe (80 kdyn) SCI followed by daily treatment with vehicle or formoterol (0.3 mg/kg, i.p.) beginning 8h after injury. The expression of pro-inflammatory cytokines/chemokines, such as IP-10, MIP-1a, MCP-1, BCA-1 and NF-κB, was increased in the injury site of vehicle-treated mice 24h post-SCI, which was ameliorated with formoterol treatment, regardless of injury severity. Thermal hyperalgesia and mechanical allodynia, as measured by Hargreaves infrared apparatus and von Frey filaments, respectively, were assessed prior to SCI and then weekly beginning 21 days post injury (DPI). While all injured mice exhibited decreased withdrawal latency following thermal stimulation compared to baseline, formoterol treatment reduced this response ~15% by 35 DPI. Vehicle-treated mice displayed significant mechanical allodynia, as evidenced by a 55% decrease in withdrawal threshold from baseline. In contrast, mice treated with formoterol maintained a consistent withdrawal time at all times tested. These data indicate that formoterol reduces inflammation post-SCI, likely contributing to mitigation of neuropathic pain, and further supporting the therapeutic potential of this treatment strategy. Significance Statement Chronic pain is a detrimental consequence of spinal cord injury (SCI). We show that treatment with the FDA-approved drug formoterol after SCI decreases injury site pro-inflammatory chemo/cytokines and alters markers of glial cell activation and infiltration. Additionally, formoterol treatment improves locomotor function and body composition, and decreases lesion volume. Finally, formoterol treatment decreased mechanical allodynia and thermal hyperalgesia post-SCI. These data are suggestive of the mechanism of formoterol-induced recovery, and further indicate its potential as a therapeutic strategy for SCI.
ABSTRACT
Breast cancer bone metastases (BMET) are incurable, primarily osteolytic, and occur most commonly in estrogen receptor-α positive (ER+) breast cancer. ER+ human breast cancer BMET modeling in mice has demonstrated an estrogen (E2)-dependent increase in tumor-associated osteolysis and bone-resorbing osteoclasts, independent of estrogenic effects on tumor proliferation or bone turnover, suggesting a possible mechanistic link between tumoral ERα-driven osteolysis and ER+ bone progression. To explore this question, inducible secretion of the osteolytic factor, parathyroid hormone-related protein (PTHrP), was utilized as an in vitro screening bioassay to query the osteolytic potential of estrogen receptor- and signaling pathway-specific ligands in BMET-forming ER+ human breast cancer cells expressing ERα, ERß, and G protein-coupled ER. After identifying genomic ERα signaling, also responsibility for estrogen's proliferative effects, as necessary and sufficient for osteolytic PTHrP secretion, in vivo effects of a genomic-only ER agonist, estetrol (E4), on osteolytic ER+ BMET progression were examined. Surprisingly, while pharmacologic effects of E4 on estrogen-dependent tissues, including bone, were evident, E4 did not support osteolytic BMET progression (vs robust E2 effects), suggesting an important role for nongenomic ER signaling in ER+ metastatic progression at this site. Because bone effects of E4 did not completely recapitulate those of E2, the relative importance of nongenomic ER signaling in tumor vs bone cannot be ascertained here. Nonetheless, these intriguing findings suggest that targeted manipulation of estrogen signaling to mitigate ER+ metastatic progression in bone may require a nuanced approach, considering genomic and nongenomic effects of ER signaling on both sides of the tumor/bone interface.
Subject(s)
Bone Neoplasms , Breast Neoplasms , Estrogen Receptor alpha , Estrogens , Signal Transduction , Bone Neoplasms/secondary , Bone Neoplasms/metabolism , Animals , Female , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Humans , Mice , Estrogens/metabolism , Estrogens/pharmacology , Estrogen Receptor alpha/metabolism , Cell Line, Tumor , Parathyroid Hormone-Related Protein/metabolism , Osteolysis/metabolism , Osteolysis/pathology , Receptors, Estrogen/metabolismABSTRACT
OBJECTIVES: Many dietary polyphenols with potential health-promoting benefits undergo hepatic conjugation and circulate as inactive glucuronides that can be cleaved by ß-glucuronidase to reform the bioactive aglycone. Although indirect evidence suggests estrogen may induce ß-glucuronidase, little is known about ß-glucuronidase regulation across women's reproductive lifespan. Correlates of serum ß-glucuronidase activity in healthy premenopausal versus postmenopausal women were therefore examined. METHODS: ß-Glucuronidase activity and C-reactive protein (CRP) were assayed in stored serum from the Women's Breast and Bone Density Study, and dual-energy x-ray absorptiometry and anthropometry assessed body composition. Participants were premenopausal (n = 133) or postmenopausal (n = 89), and Hispanic (37%) or non-Hispanic White (63%). Multivariate linear regression models tested associations between ß-glucuronidase and menopausal status, ethnicity, CRP, and body composition metrics, overall and stratified by menopausal status. RESULTS: Postmenopausal (vs premenopausal) women were older (60.4 ± 3.7 vs 44.8 ± 2.4 y) with a lower Hispanic ethnicity prevalence (27% vs 44%), and higher serum ß-glucuronidase activity (1.5 ± 0.8 vs 1.3 ± 0.5 U/L) and CRP (4.2 ± 4.4 vs 3.3 ± 4.7 mg/L). Adjusting for confounders, ß-glucuronidase was positively associated with Hispanic ethnicity, CRP, body mass index, and total fat mass (all, P < 0.01), but not menopausal status nor lean mass. Central adiposity measures were also positively associated with ß-glucuronidase with the same covariates. CONCLUSIONS: ß-Glucuronidase enzyme activity, upon which polyphenol health-related benefits may depend, is not associated with menopausal status. Future studies are required to determine clinical significance and mechanisms driving ß-glucuronidase associations with ethnicity, inflammation, and adiposity in women.
Subject(s)
Ethnicity , Postmenopause , Female , Humans , Postmenopause/physiology , Adiposity/physiology , Premenopause/physiology , Inflammation , Body Mass Index , Obesity , C-Reactive Protein/analysisABSTRACT
Globally, more than 67 million people are living with the effects of ischemic stroke. Importantly, many stroke survivors develop a chronic inflammatory response that may contribute to cognitive impairment, a common and debilitating sequela of stroke that is insufficiently studied and currently untreatable. 2-Hydroxypropyl-ß-cyclodextrin (HPßCD) is an FDA-approved cyclic oligosaccharide that can solubilize and entrap lipophilic substances. The goal of the present study was to determine whether the repeated administration of HPßCD curtails the chronic inflammatory response to stroke by reducing lipid accumulation within stroke infarcts in a distal middle cerebral artery occlusion mouse model of stroke. To achieve this goal, we subcutaneously injected young adult and aged male mice with vehicle or HPßCD 3 times per week, with treatment beginning 1 week after stroke. We evaluated mice at 7 weeks following stroke using immunostaining, RNA sequencing, lipidomic, and behavioral analyses. Chronic stroke infarct and peri-infarct regions of HPßCD-treated mice were characterized by an upregulation of genes involved in lipid metabolism and a downregulation of genes involved in innate and adaptive immunity, reactive astrogliosis, and chemotaxis. Correspondingly, HPßCD reduced the accumulation of lipid droplets, T lymphocytes, B lymphocytes, and plasma cells in stroke infarcts. Repeated administration of HPßCD also preserved NeuN immunoreactivity in the striatum and thalamus and c-Fos immunoreactivity in hippocampal regions. Additionally, HPßCD improved recovery through the protection of hippocampal-dependent spatial working memory and reduction of impulsivity. These results indicate that systemic HPßCD treatment following stroke attenuates chronic inflammation and secondary neurodegeneration and prevents poststroke cognitive decline.SIGNIFICANCE STATEMENT Dementia is a common and debilitating sequela of stroke. Currently, there are no available treatments for poststroke dementia. Our study shows that lipid metabolism is disrupted in chronic stroke infarcts, which causes an accumulation of uncleared lipid debris and correlates with a chronic inflammatory response. To our knowledge, these substantial changes in lipid homeostasis have not been previously recognized or investigated in the context of ischemic stroke. We also provide a proof of principle that solubilizing and entrapping lipophilic substances using HPßCD could be an effective strategy for treating chronic inflammation after stroke and other CNS injuries. We propose that using HPßCD for the prevention of poststroke dementia could improve recovery and increase long-term quality of life in stroke sufferers.
Subject(s)
2-Hydroxypropyl-beta-cyclodextrin/therapeutic use , Brain/drug effects , Infarction, Middle Cerebral Artery/drug therapy , Inflammation/drug therapy , Age Factors , Animals , Brain/metabolism , DNA-Binding Proteins/metabolism , Disease Models, Animal , Infarction, Middle Cerebral Artery/metabolism , Inflammation/metabolism , Male , Mice , Nerve Tissue Proteins/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Treatment OutcomeABSTRACT
The aim of this study was to test whether poststroke oral administration of a small molecule p75 neurotrophin receptor (p75NTR) modulator (LM11A-31) can augment neuronal survival and improve recovery in a mouse model of stroke. Mice were administered LM11A-31 for up to 12 weeks, beginning 1 week after stroke. Metabolomic analysis revealed that after 2 weeks of daily treatment, mice that received LM11A-31 were distinct from vehicle-treated mice by principal component analysis and had higher levels of serotonin, acetylcholine, and dopamine in their ipsilateral hemisphere. LM11A-31 treatment also improved redox homeostasis by restoring reduced glutathione. It also offset a stroke-induced reduction in glycolysis by increasing acetyl-CoA. There was no effect on cytokine levels in the infarct. At 13 weeks after stroke, adaptive immune cell infiltration in the infarct was unchanged in LM11A-31-treated mice, indicating that LM11A-31 does not alter the chronic inflammatory response to stroke at the site of the infarct. However, LM11A-31-treated mice had less brain atrophy, neurodegeneration, tau pathology, and microglial activation in other regions of the ipsilateral hemisphere. These findings correlated with improved recovery of motor function on a ladder test, improved sensorimotor and cognitive abilities on a nest construction test, and less impulsivity in an open field test. These data support small molecule modulation of the p75NTR for preserving neuronal health and function during stroke recovery. SIGNIFICANCE STATEMENT: The findings from this study introduce the p75 neurotrophin receptor as a novel small molecule target for promotion of stroke recovery. Given that LM11A-31 is in clinical trials as a potential therapy for Alzheimer's disease, it could be considered as a candidate for assessment in stroke or vascular dementia studies.
Subject(s)
Infarction, Middle Cerebral Artery/drug therapy , Isoleucine/analogs & derivatives , Morpholines/pharmacology , Neuroprotective Agents/pharmacology , Animals , Brain/drug effects , Brain/metabolism , Glutathione/metabolism , Glycolysis , Infarction, Middle Cerebral Artery/metabolism , Isoleucine/pharmacology , Isoleucine/therapeutic use , Mice , Mice, Inbred C57BL , Morpholines/therapeutic use , Neuroprotective Agents/therapeutic use , Neurotransmitter Agents/metabolism , Receptor, Nerve Growth Factor/metabolismABSTRACT
TGFß signaling promotes progression of bone-metastatic (BMET) breast cancer (BCa) cells by driving tumor-associated osteolysis, a hallmark of BCa BMETs, thus allowing for tumor expansion within bone. Turmeric-derived bioactive curcumin, enriched in bone via local enzymatic deconjugation of inactive circulating curcumin-glucuronides, inhibits osteolysis and BMET progression in human xenograft BCa BMET models by blocking tumoral TGFß signaling pathways mediating osteolysis. This is a unique antiosteolytic mechanism in contrast to current osteoclast-targeting therapeutics. Therefore, experiments were undertaken to elucidate the mechanism for curcumin inhibition of BCa TGFß signaling and the application of this finding across multiple BCa cell lines forming TGFß-dependent BMETs, including a possible role for bioactive curcumin metabolites in mediating these effects. Immunoblot analysis of TGFß signaling proteins in bone tropic human (MDA-SA, MDA-1833, MDA-2287) and murine (4T1) BCa cells revealed uniform curcumin blockade of TGFß-induced Smad activation due to down-regulation of plasma membrane associated TGFßR2 and cellular receptor Smad proteins that propagate Smad-mediated gene expression, resulting in downregulation of PTHrP expression, the osteolytic factor driving in vivo BMET progression. With the exception of early decreases in TGFßR2, inhibitory effects appeared to be mediated by oxidative metabolites of curcumin and involved inhibition of gene expression. Interestingly, while not contributing to changes in Smad-mediated TGFß signaling, curcumin caused early activation of MAPK signaling in all cell lines, including JNK, an effect possibly involving interactions with TGFßR2 within lipid rafts. Treatment with curcumin or oxidizable analogs of curcumin may have clinical relevancy in the management of TGFß-dependent BCa BMETs.
Subject(s)
Bone Neoplasms/prevention & control , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Curcumin/administration & dosage , Transforming Growth Factor beta1/metabolism , Animals , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Bone Neoplasms/secondary , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Curcumin/chemistry , Female , Humans , Mice , Oxidation-Reduction , Receptor, Transforming Growth Factor-beta Type II/genetics , Receptor, Transforming Growth Factor-beta Type II/metabolism , Signal Transduction/drug effects , Smad Proteins/genetics , Smad Proteins/metabolism , Transforming Growth Factor beta1/geneticsABSTRACT
AIM: Estrogen receptor α-positive (ER+) subtypes of breast cancer have the greatest predilection for forming osteolytic bone metastases (BMETs). Because tumor-derived factors mediate osteolysis, a possible role for tumoral ERα signaling in driving ER+ BMET osteolysis was queried using an estrogen (E2)-dependent ER+ breast cancer BMET model. METHODS: Female athymic Foxn1nu mice were inoculated with human ER+ MCF-7 breast cancer cells via the left cardiac ventricle post-E2 pellet placement, and age- and dose-dependent E2 effects on osteolytic ER+ BMET progression, as well as direct bone effects of E2, were determined. RESULTS: Osteolytic BMETs, which did not form in the absence of E2 supplementation, occurred with the same frequency in young (5-week-old) vs. skeletally mature (16-week-old) E2 (0.72 mg)-treated mice, but were larger in young mice where anabolic bone effects of E2 were greater. However, in mice of a single age and across a range of E2 doses, anabolic E2 bone effects were constant, while osteolytic ER+ BMET lesion incidence and size increased in an E2-dose-dependent fashion. Osteoclasts in ER+ tumor-bearing (but not tumor-naive) mice increased in an E2-dose dependent fashion at the bone-tumor interface, while histologic tumor size and proliferation did not vary with E2 dose. E2-inducible tumoral secretion of the osteolytic factor parathyroid hormone-related protein (PTHrP) was dose-dependent and mediated by ERα, with significantly greater levels of secretion from ER+ BMET-derived tumor cells. CONCLUSION: These results suggest that tumoral ERα signaling may contribute to ER+ BMET-associated osteolysis, potentially explaining the greater predilection for ER+ tumors to form clinically-evident osteolytic BMETs.
ABSTRACT
While tumoral Smad-mediated transforming growth factor ß (TGFß) signaling drives osteolytic estrogen receptor α-negative (ER-) breast cancer bone metastases (BMETs) in preclinical models, its role in ER+ BMETs, representing the majority of clinical BMETs, has not been documented. Experiments were undertaken to examine Smad-mediated TGFß signaling in human ER+ cells and bone-tropic behavior following intracardiac inoculation of estrogen (E2)-supplemented female nude mice. While all ER+ tumor cells tested (ZR-75-1, T47D, and MCF-7-derived) expressed TGFß receptors II and I, only cells with TGFß-inducible Smad signaling (MCF-7) formed osteolytic BMETs in vivo. Regulated secretion of PTHrP, an osteolytic factor expressed in >90% of clinical BMETs, also tracked with osteolytic potential; TGFß and E2 each induced PTHrP in bone-tropic or BMET-derived MCF-7 cells, with the combination yielding additive effects, while in cells not forming BMETs, PTHrP was not induced. In vivo treatment with 1D11, a pan-TGFß neutralizing antibody, significantly decreased osteolytic ER+ BMETs in association with a decrease in bone-resorbing osteoclasts at the tumor-bone interface. Thus, TGFß may also be a driver of ER+ BMET osteolysis. Moreover, additive pro-osteolytic effects of tumoral E2 and TGFß signaling could at least partially explain the greater propensity for ER+ tumors to form BMETs, which are primarily osteolytic.
Subject(s)
Bone Neoplasms/secondary , Breast Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Osteoclasts/pathology , Osteolysis , Receptors, Estrogen/metabolism , Transforming Growth Factor beta/metabolism , Animals , Apoptosis , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Proliferation , Female , Humans , Mice , Mice, Nude , Osteoclasts/metabolism , Receptors, Estrogen/genetics , Transforming Growth Factor beta/genetics , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
SCOPE: Curcumin prevents bone loss in resorptive bone diseases and inhibits osteoclast formation, a key process driving bone loss. Curcumin circulates as an inactive glucuronide that can be deconjugated in situ by bone's high ß-glucuronidase (GUSB) content, forming the active aglycone. Because curcumin is a common remedy for musculoskeletal disease, effects of microenvironmental changes consequent to skeletal development or disease on bone curcumin metabolism are explored. METHODS AND RESULTS: Across sexual/skeletal development or between sexes in C57BL/6 mice ingesting curcumin (500 mg kg-1 ), bone curcumin metabolism and GUSB enzyme activity are unchanged, except for >twofold higher (p < 0.05) bone curcumin-glucuronide substrate levels in immature (4-6-week-old) mice. In ovariectomized (OVX) or bone metastasis-bearing female mice, bone substrate levels are also >twofold higher. Aglycone curcumin levels tend to increase proportional to substrate such that the majority of glucuronide distributing to bone is deconjugated, including OVX mice where GUSB decreases by 24% (p < 0.01). GUSB also catalyzes deconjugation of resveratrol and quercetin glucuronides by bone, and a requirement for the aglycones for anti-osteoclastogenic bioactivity, analogous to curcumin, is confirmed. CONCLUSION: Dietary polyphenols circulating as glucuronides may require in situ deconjugation for bone-protective effects, a process influenced by bone microenvironmental changes.
Subject(s)
Bone and Bones/drug effects , Bone and Bones/metabolism , Curcumin/pharmacokinetics , Polyphenols/pharmacology , Aging , Animals , Bone Neoplasms/drug therapy , Bone Neoplasms/metabolism , Bone Neoplasms/secondary , Curcumin/administration & dosage , Curcumin/analogs & derivatives , Curcumin/metabolism , Female , Glucuronidase/metabolism , Glucuronides/pharmacokinetics , Male , Mice, Inbred C57BL , Osteogenesis/drug effects , Osteogenesis/physiology , Osteolysis/drug therapy , Osteolysis/metabolism , Osteoporosis/drug therapy , Osteoporosis/metabolism , Ovariectomy , Polyphenols/pharmacokinetics , Quercetin/pharmacologyABSTRACT
Estrogen (E2)-dependent ER+ breast cancer, the most common breast cancer subtype, is also the most likely to metastasize to bone and form osteolytic lesions. However, ER+ breast cancer bone metastasis human xenograft models in nude mice are rarely studied due to complexities associated with distinguishing possible tumoral vs. bone microenvironmental effects of E2. To address this knowledge gap, we systematically examined bone effects of E2 in developing young (4-week-old) vs. skeletally mature (15-week-old) female Foxn1nu nude mice supplemented with commercial 60-day slow-release E2 pellets and doses commonly used for ER+ xenograft models. E2 pellets (0.05-0.72 mg) were implanted subcutaneously and longitudinal changes in hind limb bones (vs. age-matched controls) were determined over 6 weeks by dual-energy X-ray absorptiometry (DXA), microCT, radiographic imaging, and histology, concurrent with assessment of serum levels of E2 and bone turnover markers. All E2 doses tested induced significant and identical increases in bone density (BMD) and volume (BV/TV) in 4-week-old mice with high bone turnover, increasing bone mineral content (BMC) while suppressing increases in bone area (BA). E2 supplementation, which caused dose-dependent changes in circulating E2 that were not sustained, also led to more modest increases in BMD and BV/TV in skeletally mature 15-week-old mice. Notably, E2-supplementation induced osteolytic osteosarcomas in a subset of mice independent of age. These results demonstrate that bone effects of E2 supplementation should be accounted for when assessing ER+ human xenograft bone metastases models.
Subject(s)
Aging/physiology , Bone and Bones/drug effects , Estradiol/administration & dosage , Osteolysis/chemically induced , Osteosarcoma/chemically induced , Absorptiometry, Photon , Animals , Bone Density/drug effects , Bone Density/physiology , Bone Neoplasms/secondary , Bone Remodeling/drug effects , Bone Remodeling/physiology , Bone and Bones/diagnostic imaging , Bone and Bones/pathology , Breast Neoplasms/pathology , Delayed-Action Preparations/administration & dosage , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Forkhead Transcription Factors/genetics , Hindlimb , Humans , Mice , Mice, Nude , Osteogenesis/drug effects , Osteogenesis/physiology , Osteolysis/pathology , Osteosarcoma/diagnostic imaging , Osteosarcoma/pathology , Receptors, Estrogen/metabolism , X-Ray Microtomography , Xenograft Model Antitumor AssaysABSTRACT
Breast cancer (BCa) bone metastases (BMETs) drive osteolysis via a feed-forward loop involving tumoral secretion of osteolytic factors (e.g., PTHrP) induced by bone-matrix-derived growth factors (e.g., TGFß). In prior experiments, turmeric-derived curcumin inhibited in vivo BMET progression and in vitro TGFß/Smad-signaling in a TGFß-stimulated PTHrP-dependent human xenograft BCa BMET model (MDA-SA cells). However, it is unclear whether curcumin or curcumin-glucuronide mediates in vivo protection since curcumin-glucuronide is the primary circulating metabolite in rodents and in humans. Thus, effects of curcumin vs. curcumin-glucuronide on Smad-dependent TGFß signaling were compared in a series of BCa cell lines forming TGFß-dependent BMET in murine models, and tissue-specific metabolism of curcumin in mice was examined by LC-MS. While curcumin inhibited TGFß-receptor-mediated Smad2/3 phosphorylation in all BCa cells studied (human MDA-SA, MDA-1833, MDA-2287 and murine 4T1 cells), curcumin-glucuronide did not. Similarly, curcumin, but not curcumin-glucuronide, blocked TGFß-stimulated secretion of PTHrP from MDA-SA and 4T1 cells. Because the predominant serum metabolite, curcumin-glucuronide, lacked bioactivity, we examined tissue-specific metabolism of curcumin in mice. Compared to serum and other organs, free curcumin (both absolute and percentage of total) was significantly increased in bone, which was also a rich source of enzymatic deglucuronidation activity. Thus, curcumin, and not curcumin-glucuronide, appears to inhibit bone-tropic BCa cell TGFß-signaling and to undergo site-specific activation (deconjugation) within the bone microenvironment. These findings suggest that circulating curcumin-glucuronide may act as a prodrug that preferentially targets bone, a process that may contribute to the bone-protective effects of curcumin and other highly glucuronidated dietary polyphenols.
Subject(s)
Bone Neoplasms/secondary , Breast Neoplasms/pathology , Curcumin/analogs & derivatives , Curcumin/pharmacology , Glucuronides/pharmacology , Smad Proteins/metabolism , Administration, Oral , Animals , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Agents, Phytogenic/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Cell Line, Tumor , Curcumin/administration & dosage , Female , Humans , Mice , Mice, Inbred C57BL , Parathyroid Hormone-Related Protein/metabolism , Signal Transduction/drug effects , Transforming Growth Factor beta/metabolismABSTRACT
Here we used mouse models of heart and brain ischemia to compare the inflammatory response to ischemia in the heart, a protein rich organ, to the inflammatory response to ischemia in the brain, a lipid rich organ. We report that ischemia-induced inflammation resolves between one and four weeks in the heart compared to between eight and 24 weeks in the brain. Importantly, we discovered that a second burst of inflammation occurs in the brain between four and eight weeks following ischemia, which coincided with the appearance of cholesterol crystals within the infarct. This second wave shares a similar cellular and molecular profile with atherosclerosis and is characterized by high levels of osteopontin (OPN) and matrix metalloproteinases (MMPs). In order to test the role of OPN in areas of liquefactive necrosis, OPN-/- mice were subjected to brain ischemia. We found that at seven weeks following stroke, the expression of pro-inflammatory proteins and MMPs was profoundly reduced in the infarct of the OPN-/- mice, although the number of cholesterol crystals was increased. OPN-/- mice exhibited faster recovery of motor function and a higher number of neuronal nuclei (NeuN) positive cells in the peri-infarct area at seven weeks following stroke. Based on these findings we propose that the brain liquefies after stroke because phagocytic cells in the infarct are unable to efficiently clear cholesterol rich myelin debris, and that this leads to the perpetuation of an OPN-dependent inflammatory response characterized by high levels of degradative enzymes.
Subject(s)
Atherosclerosis/complications , Brain Ischemia/complications , Brain/pathology , Osteopontin/pharmacology , Stroke/complications , Animals , Brain/metabolism , Disease Models, Animal , Inflammation/metabolism , Macrophages/metabolism , Male , Mice, Inbred C57BL , Neurodegenerative Diseases/pathology , Stroke/metabolismABSTRACT
The goal of this study was to determine the chronic impact of stroke on the manifestation of Alzheimer's disease (AD) related pathology and behavioral impairments in mice. To accomplish this goal, we used two distinct models. First, we experimentally induced ischemic stroke in aged wildtype (wt) C57BL/6 mice to determine if stroke leads to the manifestation of AD-associated pathological ß-amyloid (Aß) and tau in aged versus young adult wt mice. Second, we utilized a transgenic (Tg) mouse model of AD (hAPP-SL) to determine if stroke leads to the worsening of pre-existing AD pathology, as well as the development of pathology in brain regions not typically expressed in AD Tg mice. In the wt mice, there was delayed motor recovery and an accelerated development of cognitive deficits in aged mice compared to young adult mice following stroke. This corresponded with increased brain atrophy, increased cholinergic degeneration, and a focal increase of Aß in areas of axonal degeneration in the ipsilateral hemisphere of the aged animals. By contrast, in the hAPP-SL mice, we found that ischemia induced aggravated behavioral deficits in conjunction with a global increase in Aß, tau, and cholinergic pathology compared to hAPP-SL mice that underwent a sham stroke procedure. With regard to a potential mechanism, in both models, we found that the stroke-induced Aß and tau deposits co-localized with increased levels of ß-secretase 1 (BACE1), along with its substrate, neuregulin 1 (NGR1) type III, both of which are proteins integral for myelin repair. Based on these findings, we propose that the chronic sequelae of stroke may be ratcheting-up a myelin repair pathway, and that the consequent increase in BACE1 could be causing an inadvertent cleavage of its alternative substrate, AßPP, resulting in greater Aß seeding and pathogenesis.
Subject(s)
Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Dementia/metabolism , Myelin Sheath/metabolism , tau Proteins/metabolism , Age Factors , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Dementia/etiology , Disease Models, Animal , Gene Expression Regulation/genetics , Gene Expression Regulation/physiology , Humans , Male , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Motor Activity/physiology , Mutation/genetics , Plaque, Amyloid/pathology , Presenilin-1/genetics , Stroke/complicationsABSTRACT
Ginger and its extracts have been used traditionally as anti-inflammatory remedies, with a particular focus on the medicinal properties of its phenolic secondary metabolites, the gingerols. Consistent with these uses, potent anti-arthritic effects of gingerol-containing extracts were previously demonstrated by our laboratory using an experimental model of rheumatoid arthritis, streptococcal cell wall (SCW)-induced arthritis. In this study, anti-inflammatory effects of ginger's other secondary metabolites, the essential oils (GEO), which contain terpenes with reported phytoestrogenic activity, were assessed in female Lewis rats with SCW-induced arthritis. GEO (28 mg/kg/d ip) prevented chronic joint inflammation, but altered neither the initial acute phase of joint swelling nor granuloma formation at sites of SCW deposition in liver. Pharmacologic doses of 17-ß estradiol (200 or 600 µg/kg/d sc) elicited the same pattern of anti-inflammatory activity, suggesting that GEO could be acting as a phytoestrogen. However, contrary to this hypothesis, GEO had no in vivo effect on classic estrogen target organs, such as uterus or bone. En toto, these results suggest that ginger's anti-inflammatory properties are not limited to the frequently studied phenolics, but may be attributable to the combined effects of both secondary metabolites, the pungent-tasting gingerols and as well as its aromatic essential oils.
ABSTRACT
This study provides a parallel characterization of the cytokine and chemokine response to stroke in the human and mouse brain at different stages of infarct resolution. The study goal was to address the hypothesis that chronic inflammation may contribute to stroke-related dementia. We used C57BL/6 and BALB/c mice to control for strain related differences in the mouse immune response. Our data indicate that in both mouse strains, and humans, there is increased granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), interleukin-12 p70 (IL-12p70), interferon gamma-induced protein-10 (IP-10), keratinocyte chemoattractant/interleukin-8 (KC/IL-8), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), macrophage inflammatory protein-1ß (MIP-1ß), regulated on activation, normal T cell expressed and secreted (RANTES), and Tumor necrosis factor-α (TNF-α) in the infarct core during the acute time period. Nevertheless, correlation and two-way ANOVA analyses reveal that despite this substantial overlap between species, there are still significant differences, particularly in the regulation of granulocyte colony-stimulating factor (G-CSF), which is increased in mice but not in humans. In the weeks after stroke, during the stage of liquefactive necrosis, there is significant resolution of the inflammatory response to stroke within the infarct. However, CD68+ macrophages remain present, and levels of IL-6 and MCP-1 remain chronically elevated in infarcts from both mice and humans. Furthermore, there is a chronic T cell response within the infarct in both species. This response is differentially polarized towards a T helper 1 (Th1) response in C57BL/6 mice, and a T helper 2 (Th2) response in BALB/c mice, suggesting that the chronic inflammatory response to stroke may follow a different trajectory in different patients. To control for the fact that the average age of the patients used in this study was 80 years, they were of both sexes, and many had suffered from multiple strokes, we also present findings that reveal how the chronic inflammatory response to stroke is impacted by age, sex, and multiple strokes in mice. Our data indicate that the chronic cytokine and chemokine response to stroke is not substantially altered in 18-month old compared to 3-month old C57BL/6 mice, although T cell infiltration is attenuated. We found a significant correlation in the chronic cytokine response to stroke in males and females. However, the chronic cytokine response to stroke was mildly exacerbated by a recurrent stroke in both C57BL/6 and BALB/c mice.
Subject(s)
Brain Infarction/immunology , Brain/immunology , Acute Disease , Aged , Aged, 80 and over , Aging/metabolism , Aging/pathology , Animals , Brain/pathology , Brain Infarction/pathology , Chronic Disease , Female , Humans , Immunoassay , Immunohistochemistry , Infarction, Middle Cerebral Artery , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , Middle Aged , Recurrence , Sex Characteristics , Species SpecificityABSTRACT
OBJECTIVE: We sought to test the hypothesis that turmeric-derived curcuminoids limit reperfusion brain injury in an experimental model of stroke via blockade of early microvascular inflammation during reperfusion. METHODS: Male Sprague Dawley rats subjected to MCAO/R were treated with turmeric-derived curcuminoids (vs. vehicle) 1 hour prior to reperfusion (300 mg/kg ip). Neutrophil adhesion to the cerebral microcirculation and measures of neutrophil and endothelial activation were assayed during early reperfusion (0-4 hours); cerebral infarct size, edema, and neurological function were assessed at 24 hours. Curcuminoid effects on TNFα-stimulated human brain microvascular endothelial cell (HBMVEC) were assessed. RESULTS: Early during reperfusion following MCAO, curcuminoid treatment decreased neutrophil rolling and adhesion to the cerebrovascular endothelium by 76% and 67% and prevented >50% of the fall in shear rate. The increased number and activation state (CD11b and ROS) of neutrophils were unchanged by curcuminoid treatment, while increased cerebral expression of TNFα and ICAM-1, a marker of endothelial activation, were blocked by >30%. Curcuminoids inhibited NF-κB activation and subsequent ICAM-1 gene expression in HBMVEC. CONCLUSION: Turmeric-derived curcuminoids limit reperfusion injury in stroke by preventing neutrophil adhesion to the cerebrovascular microcirculation and improving shear rate by targeting the endothelium.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Curcumin/pharmacology , Endothelium, Vascular/metabolism , Neutrophil Activation/drug effects , Neutrophils/metabolism , Reperfusion Injury/metabolism , Stroke/metabolism , Animals , CD11b Antigen/metabolism , Cells, Cultured , Endothelial Cells/metabolism , Endothelial Cells/pathology , Endothelium, Vascular/pathology , Humans , Leukocyte Rolling/drug effects , Male , Neutrophils/pathology , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Reperfusion Injury/pathology , Stroke/pathologyABSTRACT
Effects of curcuminoids on breast cancer cell secretion of the bone-resorptive peptide parathyroid hormone-related protein (PTHrP) and on lytic breast cancer bone metastasis were evaluated. In vitro, transforming growth factor (TGF)-ß-stimulated PTHrP secretion was inhibited by curcuminoids (IC50 = 24 µM) in MDA-MB-231 human breast cancer cells independent of effects on cell growth inhibition. Effects on TGF-ß signaling revealed decreases in phospho-Smad2/3 and Ets-1 protein levels with no effect on p-38 MAPK-mediated TGF-ß signaling. In vivo, mice were inoculated with MDA-MB-231 cells into the left cardiac ventricle and treated ip every other day with curcuminoids (25 or 50 mg/kg) for 21 days. Osteolytic bone lesion area was reduced up to 51% (p < 0.01). Consistent with specific effects on bone osteolysis, osteoclast number at the bone-tumor interface was reduced up to 53% (p < 0.05), while tumor area within bone was unaltered. In a separate study, tumor mass in orthotopic mammary xenografts was also unaltered by treatment. These data suggest that curcuminoids prevent TGF-ß induction of PTHrP and reduce osteolytic bone destruction by blockade of Smad signaling in breast cancer cells.