ABSTRACT
COVID-19, caused by SARS-CoV-2, affects neuronal cells, causing several symptoms such as memory loss, anosmia and brain inflammation. Curcuminoids (Me08 e Me23) and curcumin (CUR) are derived from Curcuma Longa extract (EXT). Many therapeutic actions have been linked to these compounds, including antiviral action. Given the severe implications of COVID-19, especially within the central nervous system, our study aims to shed light on the therapeutic potential of curcuminoids against SARS-CoV-2 infection, particularly in neuronal cells. Here, we investigated the effects of CUR, EXT, Me08 and Me23 in human neuroblastoma SH-SY5Y. We observed that Me23 significantly decreased the expression of plasma membrane-associated transmembrane protease serine 2 (TMPRSS2) and TMPRSS11D, consequently mitigating the elevated ROS levels induced by SARS-CoV-2. Furthermore, Me23 exhibited antioxidative properties by increasing NRF2 gene expression and restoring NQO1 activity following SARS-CoV-2 infection. Both Me08 and Me23 effectively reduced SARS-CoV-2 replication in SH-SY5Y cells overexpressing ACE2 (SH-ACE2). Additionally, all of these compounds demonstrated the ability to decrease proinflammatory cytokines such as IL-6, TNF-α, and IL-17, while Me08 specifically reduced INF-γ levels. Our findings suggest that curcuminoid Me23 could serve as a potential agent for mitigating the impact of COVID-19, particularly within the context of central nervous system involvement.
Subject(s)
Anti-Inflammatory Agents , Antioxidants , Antiviral Agents , COVID-19 Drug Treatment , Curcumin , SARS-CoV-2 , Humans , Curcumin/pharmacology , Curcumin/analogs & derivatives , Antioxidants/pharmacology , Antiviral Agents/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Anti-Inflammatory Agents/pharmacology , Cell Line, Tumor , Curcuma/chemistry , Serine Endopeptidases/metabolism , COVID-19/virology , COVID-19/metabolism , Reactive Oxygen Species/metabolism , NF-E2-Related Factor 2/metabolism , Plant Extracts/pharmacology , Cytokines/metabolism , NAD(P)H Dehydrogenase (Quinone)/metabolism , Neurons/drug effects , Neurons/metabolism , Neurons/virologyABSTRACT
'On-a-chip' technology advances the development of physiologically relevant organ-mimicking architecture by integrating human cells into three-dimensional microfluidic devices. This method also establishes discrete functional units, faciliting focused research on specific organ components. In this study, we detail the development and assessment of a convoluted renal proximal tubule-on-a-chip (PT-on-a-chip). This platform involves co-culturing Renal Proximal Tubule Epithelial Cells (RPTEC) and Human Umbilical Vein Endothelial Cells (HUVEC) within a polydimethylsiloxane microfluidic device, crafted through a combination of 3D printing and molding techniques. Our PT-on-a-chip significantly reduced high glucose level, exhibited albumin uptake, and simulated tubulopathy induced by amphotericin B. Remarkably, the RPTEC:HUVEC co-culture exhibited efficient cell adhesion within 30 min on microchannels functionalized with plasma, 3-aminopropyltriethoxysilane, and type-I collagen. This approach significantly reduced the required incubation time for medium perfusion. In comparison, alternative methods such as plasma and plasma plus polyvinyl alcohol were only effective in promoting cell attachment to flat surfaces. The PT-on-a-chip holds great promise as a valuable tool for assessing the nephrotoxic potential of new drug candidates, enhancing our understanding of drug interactions with co-cultured renal cells, and reducing the need for animal experimentation, promoting the safe and ethical development of new pharmaceuticals.
Subject(s)
Epithelial Cells , Kidney Tubules, Proximal , Animals , Humans , Human Umbilical Vein Endothelial Cells , Coculture Techniques , Kidney Tubules, Proximal/metabolism , Lab-On-A-Chip DevicesABSTRACT
COVID-19, along with most respiratory diseases in the medical field, demonstrates significant ability to take its toll on global population. There is a particular difficulty in studying these conditions, which stems especially from the short supply of in vitro models for detailed investigation, the specific therapeutic knowledge required for disease scrutinization and the occasional need of BSL-3 [Biosafety Level 3] laboratories for research. Based on this, the process of drug development is hampered to a great extent. In the scenario of COVID-19, this difficulty is even more substantial on account of the current undefinition regarding the exact role of the ACE2 [Angiotensin-converting enzyme 2] receptor upon SARS-CoV-2 kinetics in human cells and the great level of demand in the investigation process of ACE2, which usually requires the laborious and ethically complicated usage of transgenic animal models overexpressing the receptor. Moreover, the rapid progression of the aforementioned diseases, especially COVID-19, poses a crucial necessity for adequate therapeutic solutions emergence. In this context, the work herein presented introduces a groundbreaking set of 3D models, namely spheroids and MatriWell cell culture inserts, whose remarkable ability to mimic the in vivo environment makes them highly suitable for respiratory diseases investigation, particularly SARS-CoV-2 infection. Using MatriWells, we developed an innovative platform for COVID-19 research: a pulmonary air-liquid interface [ALI] associated with endothelial (HUVEC) cells. Infection studies revealed that pulmonary (BEAS-2B) cells in the ALI reached peak viral load at 24h and endothelial cells, at 48h, demonstrating lung viral replication and subsequent hematogenous dissemination, which provides us with a unique and realistic framework for studying COVID-19. Simultaneously, the spheroids were used to address the understudied ACE2 receptor, aiming at a pronounced process of COVID-19 investigation. ACE2 expression not only increased spheroid diameter by 20% (p<0.001) and volume by 60% (p≤0.0001) but also led to a remarkable 640-fold increase in intracellular viral load (p≤0.01). The previously mentioned finding supports ACE2 as a potential target for COVID-19 treatment. Lastly, we observed a higher viral load in the MatriWells compared to spheroids (150-fold, p<0.0001), suggesting the MatriWells as a more appropriate approach for COVID-19 investigation. By establishing an advanced method for respiratory tract conditions research, this work paves the way toward an efficacious process of drug development, contributing to a change in the course of respiratory diseases such as COVID-19.
ABSTRACT
Alzheimer's disease (AD) is the most prevalent aging-associated neurodegenerative disease, with a higher incidence in women than men. There is evidence that sex hormone replacement therapy, particularly estrogen, reduces memory loss in menopausal women. Neurofibrillary tangles are associated with tau protein aggregation, a characteristic of AD and other tauopathies. In this sense, autophagy is a promising cellular process to remove these protein aggregates. This study evaluated the autophagy mechanisms involved in neuroprotection induced by 17ß-estradiol (E2) in a Tet-On inducible expression tauopathy cell model (EGFP-tau WT or with the P301L mutation, 0N4R isoform). The results indicated that 17ß-estradiol induces autophagy by activating AMPK in a concentration-dependent manner, independent of mTOR signals. The estrogen receptor α (ERα) agonist, PPT, also induced autophagy, while the ERα antagonist, MPP, substantially attenuated the 17ß-estradiol-mediated autophagy induction. Notably, 17ß-estradiol increased LC3-II levels and phosphorylated and total tau protein clearance in the EGFP-tau WT cell line but not in EGPF-tau P301L. Similar results were observed with E2-BSA, a plasma membrane-impermeable estrogen, suggesting membrane ERα involvement in non-genomic estrogenic pathway activation. Furthermore, 17ß-estradiol-induced autophagy led to EGFP-tau protein clearance. These results demonstrate that modulating autophagy via the estrogenic pathway may represent a new therapeutic target for treating AD.
Subject(s)
Alzheimer Disease , Neurodegenerative Diseases , Tauopathies , AMP-Activated Protein Kinases , Autophagy , Estradiol/pharmacology , Estrogen Receptor alpha/metabolism , Estrogens/pharmacology , Female , Humans , Male , Protein Aggregates , Receptors, Estrogen , TOR Serine-Threonine Kinases , tau Proteins/metabolismABSTRACT
Gender-bias in COVID-19 severity has been suggested by clinical data. Experimental data in cell and animal models have demonstrated the role of sex hormones, particularly estrogens, in viral infections such as in COVID-19. SARS-CoV-2 uses ACE2 as a receptor to recognize host cells, and the protease TMPRSS2 for priming the Spike protein, facilitating virus entry into cells. However, the involvement of estrogenic receptors in SARS-CoV-2 infection are still being explored. Thus, in order to investigate the role of estrogen and its receptors in COVID-19, the estrogen receptors ERα, ERß and GPER1 were overexpressed in bronchial BEAS-2B cell, and then infected with SARS-CoV-2. Interestingly, the levels of ACE2 and TMPRSS2 mRNA were higher in SARS-CoV-2-infected cells, but no difference was observed in cells with estrogen receptors overexpression. GPER1 can be involved in virus infection or replication, since its higher levels reduces SARS-CoV-2 load. On the other hand, pharmacological antagonism of GPER1 enhanced viral load. Those data suggest that GPER1 has an important role in SARS-CoV-2 infection.
Subject(s)
COVID-19 , Animals , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Receptors, Estrogen , Estrogen Receptor beta , Estrogen Receptor alpha , Peptidyl-Dipeptidase A/metabolism , RNA, Messenger/genetics , EstrogensABSTRACT
AIMS: This study evaluated SARS-CoV-2 replication in human cell lines derived from various tissues and investigated molecular mechanisms related to viral infection susceptibility and replication. MAIN METHODS: SARS-CoV-2 replication in BEAS-2B and A549 (respiratory tract), HEK-293 T (kidney), HuH7 (liver), SH-SY5Y (brain), MCF7 (breast), Huvec (endothelial) and Caco-2 (intestine) was evaluated by RT-qPCR. Concomitantly, expression levels of ACE2 (Angiotensin Converting Enzyme) and TMPRSS2 were assessed through RT-qPCR and western blot. Proteins related to autophagy and mitochondrial metabolism were monitored in uninfected cells to characterize the cellular metabolism of each cell line. The effect of ACE2 overexpression on viral replication in pulmonary cells was also investigated. KEY FINDINGS: Our data show that HuH7, Caco-2 and MCF7 presented a higher viral load compared to the other cell lines. The increased susceptibility to SARS-CoV-2 infection seems to be associated not only with the differential levels of proteins intrinsically related to energetic metabolism, such as ATP synthase, citrate synthase, COX and NDUFS2 but also with the considerably higher TMPRSS2 mRNA expression. The two least susceptible cell types, BEAS-2B and A549, showed drastically increased SARS-CoV-2 replication capacity when ACE2 was overexpressed. These modified cell lines are relevant for studying SARS-CoV-2 replication in vitro. SIGNIFICANCE: Our data not only reinforce that TMPRSS2 expression and cellular energy metabolism are important molecular mechanisms for SARS-CoV-2 infection and replication, but also indicate that HuH7, MCF7 and Caco-2 are suitable models for mechanistic studies of COVID-19. Moreover, pulmonary cells overexpressing ACE2 can be used to understand mechanisms associated with SARS-CoV-2 replication.
Subject(s)
COVID-19 , Neuroblastoma , Adenosine Triphosphate , Angiotensin-Converting Enzyme 2/genetics , Autophagy , Caco-2 Cells , Citrate (si)-Synthase , HEK293 Cells , Humans , Peptidyl-Dipeptidase A/metabolism , RNA, Messenger/genetics , SARS-CoV-2ABSTRACT
COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been considered a public health emergency, extensively investigated by researchers. Accordingly, the respiratory tract has been the main research focus, with some other studies outlining the effects on the neurological, cardiovascular, and renal systems. However, concerning SARS-CoV-2 outcomes on skeletal muscle, scientific evidence is still not sufficiently strong to trace, treat and prevent possible muscle impairment due to the COVID-19. Simultaneously, there has been a considerable amount of studies reporting skeletal muscle damage in the context of COVID-19. Among the detrimental musculoskeletal conditions associated with the viral infection, the most commonly described are sarcopenia, cachexia, myalgia, myositis, rhabdomyolysis, atrophy, peripheral neuropathy, and Guillain-Barré Syndrome. Of note, the risk of developing sarcopenia during or after COVID-19 is relatively high, which poses special importance to the condition amid the SARS-CoV-2 infection. The yet uncovered mechanisms by which musculoskeletal injury takes place in COVID-19 and the lack of published methods tailored to study the correlation between COVID-19 and skeletal muscle hinder the ability of healthcare professionals to provide SARS-CoV-2 infected patients with an adequate treatment plan. The present review aims to minimize this burden by both thoroughly exploring the interaction between COVID-19 and the musculoskeletal system and examining the cutting-edge 3D cell culture techniques capable of revolutionizing the study of muscle dynamics.
ABSTRACT
AIMS: The purpose of this study was to investigate the effect of PPRP (pure PRP) and LPRP (PRP with leukocytes) on recovery from limb ischemia and on expression of growth factors involved in angiogenesis, myogenesis and fibrogenesis. MATERIAL AND METHODS: PPRP and LPRP prepared by centrifugation were added to cultures of C2C12 and NIH3T3 cells (1 or 10% PRPs) to evaluate alterations in cell metabolism and expression of growth factors by MTT, ELISA and RT-qPCR, respectively. To evaluate in vivo regenerative effects, PRPs were injected into the ischemic limbs of BALB/c mice and muscle mass/strength and histomorphometry were evaluated after 30 days. KEY FINDINGS: Mice treated with PRPs after limb ischemia showed an increase in the size of myofibers and muscle strength, reduced fibrosis and adipocytes, and decreased capillary density and necrosis scores compared to untreated mice. In cell culture, serum deprivation reduced the viability of C2C12 and NIH3T3 cells to about 50%, but the addition of 1% PRPs completely recovered this loss. Both PRPs, downregulated most of the tested genes; however, angiogenic gene Vegfa in C2C12 and the fibrogenic genes Col1a1 and Col3a1 in NIH3T3 cells were upregulated by LPRP. SIGNIFICANCE: PPRP and LPRP had similar effects in regulation of genes involved in angiogenesis, myogenesis and fibrogenesis. However, the presence of leucocytes did not significantly affect regenerative activities of PRP in the ischemic limb.
Subject(s)
Hindlimb/physiopathology , Ischemia/physiopathology , Platelet-Rich Plasma/metabolism , Regeneration/physiology , Animals , Cell Survival , Gene Expression Regulation , Mice , Mice, Inbred C57BL , Muscle, Skeletal/pathology , NIH 3T3 CellsABSTRACT
The COVID-19 has originated from Wuhan, China, in December 2019 and has been affecting the public health system, society, and economy in an unheard-of manner. There is no specific treatment or vaccine available for COVID-19. Previous data showed that men are more affected than women by COVID-19, then we hypothesized whether sex hormones could be protecting the female organism against the infection. VERO E6 cells have been commonly used as in vitro model for SARS-CoV-2 infection. In our experimental approach, we have treated VERO E6 cells with 17ß-estradiol to evaluate the modulation of SARS-CoV-2 infection in this cell line. Here we demonstrated that estrogen protein receptors ERα, ERß, and GPER1 are expressed by VERO E6 cells and could be used to study the effects of this steroid hormone. Previous and 24-hours post-infection, cells treated with 17ß-estradiol revealed a reduction in the viral load. Afterward, we found that SARS-CoV-2 infection per se results in ACE2 and TMPRSS2 increased gene expression in VERO E6-cell, which could be generating a cycle of virus infection in host cells. The estrogen treatment reduces the levels of the TMPRSS2, which are involved with SARS-CoV-2 infectiveness capacity, and hence, reducing the pathogenicity/genesis. These data suggest that estrogen could be a potential therapeutic target promoting cell protection against SARS-CoV-2. This opens new possibilities for further studies on 17ß-estradiol in human cell lines infected by SARS-CoV-2 and at least in part, explain why men developed a more severe COVID-19 compared to women.
Subject(s)
Antiviral Agents/pharmacology , COVID-19 Drug Treatment , Estradiol/pharmacology , SARS-CoV-2/drug effects , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/metabolism , COVID-19/virology , Chlorocebus aethiops , Host-Pathogen Interactions , Receptors, Virus/genetics , Receptors, Virus/metabolism , SARS-CoV-2/pathogenicity , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Vero CellsABSTRACT
BACKGROUND: After traumatic skeletal muscle injury, muscle healing is often incomplete and produces extensive fibrosis. The sequence of M1 and M2 macrophage accumulation and the duration of each subtype in the injured area may help to direct the relative extent of fibrogenesis and myogenesis during healing. We hypothesized that increasing the number of M1 macrophages early after traumatic muscle injury would produce more cellular and molecular substrates for myogenesis and fewer substrates for fibrosis, leading to better muscle healing. METHODS: To test this hypothesis, we transfected skeletal muscle with a plasmid vector to transiently express GM-CSF shortly after injury to drive the polarization of macrophages towards the M1 subset. C57BL/6 mouse tibialis anterior (TA) muscles were injured by contusion and electroporated with uP-mGM, which is a plasmid vector that transiently expresses GM-CSF. Myogenesis, angiogenesis, and fibrosis were evaluated by histology, immunohistochemistry, and RT-qPCR; subpopulations of macrophages by flow cytometry; and muscle functioning by the maximum running speed on the treadmill and the recovery of muscle mass. RESULTS: Muscle injury increased the number of local M1-like macrophages and decreased the number of M2-like macrophages on day 4, and uP-mGM treatment enhanced this variation. uP-mGM treatment decreased TGF-ß1 protein expression on day 4, and the Sirius Red-positive area decreased from 35.93 ± 15.45% (no treatment) to 2.9% ± 6.5% (p < 0.01) on day 30. uP-mGM electroporation also increased Hgf, Hif1α, and Mtor gene expression; arteriole density; and muscle fiber number during regeneration. The improvement in the quality of the muscle tissue after treatment with uP-mGM affected the increase in the TA muscle mass and the maximum running speed on a treadmill. CONCLUSION: Collectively, our data show that increasing the number of M1-like macrophages immediately after traumatic muscle injury promotes muscle recovery with less fibrosis, and this can be achieved by the transient expression of GM-CSF.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor , Macrophages , Animals , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Mice , Mice, Inbred C57BL , Muscle, Skeletal , Wound HealingABSTRACT
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has provoked major stresses on the health-care systems of several countries, and caused the death of more than a quarter of a million people globally, mainly in the elderly population with preexisting pathologies. Previous studies with coronavirus (SARS-CoV) point to gender differences in infection and disease progression with increased susceptibility in male patients, indicating that estrogens may be associated with physiological protection against the coronavirus. Therefore, the objectives of this work are threefold. First, we aim to summarize the SARS-CoV-2 infection pathway and the roles both the virus and patient play in COVID-19 (Coronavirus disease 2019) progression, clinical symptomatology, and mortality. Second, we detail the effect estrogen has on viral infection and host infection response, including its role in both the regulation of key viral receptor expression and the mediation of inflammatory activity. Finally, we describe how ERs (estrogen receptors) and RAGE (receptor for advanced glycation end-products) play a critical role in metabolic pathways, which we envisage could maintain a close interplay with SARS-CoV and COVID-19 mortality rates, despite a current lack of research directly determining how. Taken together, we present the current state of the field regarding SARS-CoV-2 research and illuminate where research is needed to better define the role both estrogen and metabolic comorbidities have in the COVID-19 disease state, which can be key in screening potential therapeutic options as the search for effective treatments continue.
Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/epidemiology , Coronavirus Infections/pathology , Pneumonia, Viral/epidemiology , Pneumonia, Viral/pathology , Age Factors , Angiotensin-Converting Enzyme 2 , Animals , Antigens, Neoplasm/metabolism , COVID-19 , Coronavirus Infections/immunology , Coronavirus Infections/metabolism , Disease Susceptibility , Estrogens/metabolism , Female , Humans , Lung/pathology , Male , Mitogen-Activated Protein Kinases/metabolism , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/immunology , Pneumonia, Viral/metabolism , Receptors, Estrogen/metabolism , SARS-CoV-2 , Sex Factors , Signal TransductionABSTRACT
Considering that female sexual hormones may modulate the inflammatory response and also exhibit direct effects on the cells of the immune system, herein, we intend to discuss the sex differences and the role of estradiol in modulating the lung and systemic inflammatory response, focusing on its possible application as a treatment modality for SARS-CoV-2 patients. COVID-19 patients develop severe hypoxemia early in the course of the disease, which is silent most of the time. Small fibrinous thrombi in pulmonary arterioles and a tumefaction of endothelial were observed in the autopsies of fatal COVID-19 cases. Studies showed that the viral infection induces a vascular process in the lung, which included vasodilation and endothelial dysfunction. Further, the proportions of CD4+ T and CD8+ T lymphocytes were strongly reduced in patients with severe SARS-CoV-2 infection. Estradiol is connected with CD4+ T cell numbers and increases T-reg cell populations, affecting immune responses to infection. It is known that estradiol exerts a protective effect on endothelial function, activating the generation of nitric oxide (NO) via endothelial nitric oxide synthase. Estrogen attenuates the vasoconstrictor response to various stimuli and induces vasodilation in the pulmonary vasculature during stress situations like hypoxia. It exerts a variety of rapid actions, which are initiated after its coupling with membrane receptors, which in turn, may positively modulate vascular responses in pulmonary disease and help to maintain microvascular flow. Direct and indirect mechanisms underlying the effects of estradiol were investigated, and the results point to a possible protective effect of estradiol against COVID-19, indicating that it may be considered as an adjuvant therapeutic element for the treatment of patients affected by the novel coronavirus.
Subject(s)
Betacoronavirus , Coronavirus Infections/therapy , Estradiol/therapeutic use , Immunity, Innate , Inflammation/virology , Pneumonia, Viral/therapy , Animals , COVID-19 , Female , Humans , Inflammation/drug therapy , Male , Pandemics , Rats , SARS-CoV-2 , Sex FactorsABSTRACT
Considering that female sexual hormones may modulate the inflammatory response and also exhibit direct effects on the cells of the immune system, herein, we intend to discuss the sex differences and the role of estradiol in modulating the lung and systemic inflammatory response, focusing on its possible application as a treatment modality for SARS-CoV-2 patients. COVID-19 patients develop severe hypoxemia early in the course of the disease, which is silent most of the time. Small fibrinous thrombi in pulmonary arterioles and a tumefaction of endothelial were observed in the autopsies of fatal COVID-19 cases. Studies showed that the viral infection induces a vascular process in the lung, which included vasodilation and endothelial dysfunction. Further, the proportions of CD4+ T and CD8+ T lymphocytes were strongly reduced in patients with severe SARS-CoV-2 infection. Estradiol is connected with CD4+ T cell numbers and increases T-reg cell populations, affecting immune responses to infection. It is known that estradiol exerts a protective effect on endothelial function, activating the generation of nitric oxide (NO) via endothelial nitric oxide synthase. Estrogen attenuates the vasoconstrictor response to various stimuli and induces vasodilation in the pulmonary vasculature during stress situations like hypoxia. It exerts a variety of rapid actions, which are initiated after its coupling with membrane receptors, which in turn, may positively modulate vascular responses in pulmonary disease and help to maintain microvascular flow. Direct and indirect mechanisms underlying the effects of estradiol were investigated, and the results point to a possible protective effect of estradiol against COVID-19, indicating that it may be considered as an adjuvant therapeutic element for the treatment of patients affected by the novel coronavirus.
Subject(s)
Humans , Animals , Male , Female , Rats , Pneumonia, Viral/therapy , Coronavirus Infections/therapy , Estradiol/therapeutic use , Betacoronavirus , Immunity, Innate , Inflammation/virology , Sex Factors , Pandemics , SARS-CoV-2 , COVID-19 , Inflammation/drug therapyABSTRACT
Dystrophin deficiency caused by mutations of the related gene leads to muscle wasting in Duchenne muscular dystrophy (DMD). Some patients with DMD also present with intellectual disability and various degrees of neurological disorders, which have been related to a decreased number of postsynaptic gamma-aminobutyric acid type A receptors (GABAARs) in the hippocampus (HPC) and cerebellum (CBL). The aim of this study was to examine the relevance of dystrophin in the presynaptic GABAergic function in brain regions in which this protein is normally abundant. [3H]-GABA release, induced by nicotinic receptor (nAChR) activation or K+ depolarization, and [3H]-GABA uptake were determined using synaptosomes extracted from the cortex (CTX), HPC, and CBL of littermate control and mdx mice. Superfusion of the synaptosomes with nicotine or high K+ solutions led to a concentration-dependent and Ca2+-dependent [3H]-GABA release in control and mdx synaptosomes. [3H]-GABA release induced by 10⯵M nicotine in mdx CBL synaptosomes was 47% less than that in control mice. K+-induced [3H]-GABA release did not differ between control and mdx synaptosomes. α7-containing and ß2-containing nAChRs were involved in nicotine-induced [3H]-GABA release in control and mdx synaptosomes. Kinetic analysis of [3H]-GABA uptake in mdx CBL synaptosomes showed a reduced (50%) half-maximal uptake time (t1/2) and increased (44%) rate of [3H]-GABA uptake (Vmax) compared to controls. The apparent transporter affinity (Km) for GABA was not altered. Our findings show that dystrophin deficiency in mdx mice is associated with significant changes in the release and uptake of GABA in the CBL. These presynaptic alterations may be related to the reported decrease in postsynaptic GABAAR in the same brain region. The results indicate possible dysfunction of GABAergic synapses associated with dystrophin deficiency in the CBL, which may contribute to the cognitive and neurobehavioral disorders in mdx mice and patients with DMD.
Subject(s)
Cerebellum/metabolism , Dystrophin/deficiency , Muscular Dystrophy, Duchenne/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Cerebellum/ultrastructure , Dystrophin/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Muscular Dystrophy, Duchenne/geneticsABSTRACT
α-Synuclein is the major component of neuronal cytoplasmic aggregates called Lewy bodies, the main pathological hallmark of Parkinson disease. Although neurons are the predominant cells expressing α-synuclein in the brain, recent studies have demonstrated that primary astrocytes in culture also express α-synuclein and regulate α-synuclein trafficking. Astrocytes have a neuroprotective role in several detrimental brain conditions; we therefore analyzed the effects of the overexpression of wild-type α-synuclein and its A30P and A53T mutants on autophagy and apoptosis. We observed that in immortalized astrocyte cell lines, overexpression of α-synuclein proteins promotes the decrease of LC3-II and the increase of p62 protein levels, suggesting the inhibition of autophagy. When these cells were treated with rotenone, there was a loss of mitochondrial membrane potential, especially in cells expressing mutant α-synuclein. The level of this decrease was related to the toxicity of the mutants because they show a more intense and sustained effect. The decrease in autophagy and the mitochondrial changes in conjunction with parkin expression levels may sensitize astrocytes to apoptosis.
Subject(s)
Apoptosis/physiology , Astrocytes/metabolism , Autophagy/physiology , alpha-Synuclein/biosynthesis , Animals , Astrocytes/pathology , Cell Line, Transformed , Cells, Cultured , Female , Gene Expression , Male , Rats , Rats, Wistar , alpha-Synuclein/geneticsABSTRACT
Regeneration of injured skeletal muscles is affected by fibrosis, which can be improved by the administration of angiotensin II (AngII) receptor (ATR) blockers in normotensive animals. However, the role of ATR in skeletal muscle fibrosis in hypertensive organisms has not been investigated yet. The tibialis anterior (TA) muscle of spontaneously hypertensive (SHR) and Wistar rats (WR) were lacerated and a lentivector encoding a microRNA targeting AngII receptor type 1 (At1) (Lv-mirAT1a) or control (Lv-mirCTL) was injected. The TA muscles were collected after 30 days to evaluate fibrosis by histology and gene expression by real-time quantitative PCR (RT-qPCR) and Western blot. SHR's myoblasts were analyzed by RT-qPCR, 48 h after transduction. In the SHR's TA, AT1 protein expression was 23.5-fold higher than in WR without injury, but no difference was observed in the angiotensin II receptor type 2 (AT2) protein expression. TA laceration followed by suture (LS) produced fibrosis in the SHR (23.3±8.5%) and WR (7.9±1.5%). Lv-mirAT1 treatment decreased At1 gene expression in 50% and reduced fibrosis to 7% 30 days after. RT-qPCR showed that reduction in At1 expression is due to downregulation of the At1a but not of the At1b. RT-qPCR of myoblasts from SHR transduced with Lv-mirAT1a showed downregulation of the Tgf-b1, Tgf-b2, Smad3, Col1a1, and Col3a1 genes by mirAT1a. In vivo and in vitro studies indicate that hypertension overproduces skeletal muscle fibrosis, and AngII-AT1a signaling is the main pathway of fibrosis in SHR. Moreover, muscle fibrosis can be treated specifically by in loco injection of Lv-mirAT1a without affecting other organs.
Subject(s)
MicroRNAs/genetics , Muscle, Skeletal/pathology , Receptors, Angiotensin/genetics , Animals , Down-Regulation , Fibrosis , Gene Expression Profiling , Rats , Rats, Inbred SHR , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The subventricular zone (SVZ) of the adult mammalian brain hosts full potential neural stem cells (NSCs). NSCs are able to respond to extracellular signals in the brain, amplifying the pool of progenitor cells and giving rise to neuroblasts that show ability to migrate towards an injury site. These signals can come from vascular system, cerebrospinal fluid, glial cells, or projections of neurons in adjoining regions. CXCL12, a chemokine secreted after brain injury, reaches the SVZ in a gradient manner and drives neuroblasts towards the lesion area. Among many other molecules, matrix metalloproteinase 2 and 9 (MMP-2/9) are also released during brain injury. MMP-2/9 can cleave CXCL12 generating a new molecule, CXCL12(5-67), and its effects on NSCs viability is not well described. Here we produced recombinant CXCL12 and CXCL12(5-67) and evaluated their effect in murine adult NSCs migration and survival in vitro. We showed CXCL12(5-67) does not promote NSCs migration, but does induce cell death. The NSC death induced by CXCL12(5-67) involves caspases 9 and 3/7 activation, implying the intrinsic apoptotic pathway in this phenomenon. Our evidences in vitro make CXCL12(5-67) and its receptor potential candidates for brain injuries and neurodegeneration studies.
Subject(s)
Chemokine CXCL12/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Amino Acid Sequence , Animals , Apoptosis/physiology , Base Sequence , Cell Differentiation/physiology , Cell Movement/physiology , Chemokine CXCL12/pharmacology , Chemotaxis/drug effects , Female , HEK293 Cells , Humans , Jurkat Cells , Mice , Mice, Inbred C57BL , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Receptors, CXCR4/metabolism , Recombinant Proteins/pharmacologyABSTRACT
BACKGROUND: It is well known that platelet-rich plasma (PRP) preparations are not the same and that not all preparations include white blood cells, but the part that leukocytes play on the healing role of PRP is still unknown. PURPOSE: The primary aim of this study was to evaluate the influence of leukocytes in different PRP preparations with a special emphasis on growth factor concentrations. The secondary aim was to evaluate the influence of PRP on muscle healing. STUDY DESIGN: Controlled laboratory study. METHODS: Two PRP preparation procedures were evaluated. Blood fractions were stained with Rapid Panoptic, and growth factors (transforming growth factor beta 1 [TGF-ß1], vascular endothelial growth factor [VEGF], insulin-like growth factor [IGF], epidermal growth factor [EGF], hepatocyte growth factor [HGF], and platelet-derived growth factor [PDGF]) were quantified by enzyme-linked immunosorbent assay. Western blotting analysis was performed for Fms-related tyrosine kinase 1 (Flt-1). A muscle contusion injury was created and treated with PRP at different time points. RESULTS: Leukocytes were the main source of VEGF, and all other growth factors measured had a higher concentration in the preparations that included the buffy coat and consequently had a higher concentration of white blood cells. Flt-1 was also found in platelet-poor plasma (PPP). There were higher concentrations of PDGF and HGF in the preparations that encompassed the buffy coat. A PRP injection 7 days after the injury provided significantly increased exercise performance and decreased the fibrotic area when compared with other PRP-treated groups. CONCLUSION: VEGF is only present in PRP's buffy coat, while Flt-1 is present in PPP. A PRP injection 7 days after an injury resulted in improved exercise performance. CLINICAL RELEVANCE: The presence of Flt-1 in PRP provides yet another explanation for results described in the literature after a PRP injection. This information is relevant for selecting the best PRP for each type of injury.
Subject(s)
Contusions/drug therapy , Leukocytes/metabolism , Muscles/injuries , Platelet-Rich Plasma/chemistry , Vascular Endothelial Growth Factor Receptor-1/metabolism , Animals , Contusions/metabolism , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Humans , Male , Mice , Mice, Inbred C57BL , Muscles/metabolism , Muscles/physiopathology , Platelet-Derived Growth Factor/metabolism , Transforming Growth Factor beta1/metabolism , Vascular Endothelial Growth Factor A/metabolism , Wound Healing/drug effectsABSTRACT
Glioblastoma (GBM) is an infiltrative tumor that is difficult to eradicate. Treating GBM with mesenchymal stem cells (MSCs) that have been modified with the HSV-Tk suicide gene has brought significant advances mainly because MSCs are chemoattracted to GBM and kill tumor cells via a bystander effect. To use this strategy, abundantly present adipose-tissue-derived mesenchymal stem cells (AT-MSCs) were evaluated for the treatment of GBM in mice. AT-MSCs were prepared using a mechanical protocol to avoid contamination with animal protein and transduced with HSV-Tk via a lentiviral vector. The U-87 glioblastoma cells cultured with AT-MSC-HSV-Tk died in the presence of 25 or 50 µM ganciclovir (GCV). U-87 glioblastoma cells injected into the brains of nude mice generated tumors larger than 3.5 mm2 after 4 weeks, but the injection of AT-MSC-HSV-Tk cells one week after the U-87 injection, combined with GCV treatment, drastically reduced tumors to smaller than 0.5 mm2. Immunohistochemical analysis of the tumors showed the presence of AT-MSC-HSV-Tk cells only within the tumor and its vicinity, but not in other areas of the brain, showing chemoattraction between them. The abundance of AT-MSCs and the easier to obtain them mechanically are strong advantages when compared to using MSCs from other tissues.
Subject(s)
Adipose Tissue/metabolism , Glioblastoma/metabolism , Mesenchymal Stem Cells/enzymology , Simplexvirus/genetics , Thymidine Kinase/biosynthesis , Transduction, Genetic , Viral Proteins/biosynthesis , Adipose Tissue/pathology , Animals , Bystander Effect/drug effects , Cell Line, Tumor , Female , Ganciclovir/pharmacology , Glioblastoma/pathology , Glioblastoma/therapy , Humans , Mesenchymal Stem Cells/pathology , Mice , Mice, Nude , Simplexvirus/enzymology , Thymidine Kinase/genetics , Viral Proteins/geneticsABSTRACT
Skeletal muscle injury and healing are multifactorial processes, involving three steps of healing: (1) degeneration and inflammation, (2) regeneration, and (3) fibrosis. Fibrous tissue hinders the muscle's complete recovery and current therapies fail in achieving total muscle recovery. Gene and cell therapy (or both) are potential future treatments for severe muscular injuries. Stem cells' properties associated with growth factors or/and cytokines can improve muscle healing and permit long-term recovery.