Criopreservação de células-tronco mesenquimais do tecido adiposo: Uma alternativa para lipoenxertia seriada na reconstrução mamária / Cryopreservation of adipose tissue-derived mesenchymal stem cells: An alternative to serial fat grafting in breast reconstruction

Introdução: A lipoenxertia é um enxerto autólogo de células do tecido celular subcutâneo, que pode ser utilizada como técnica complementar na reconstrução mamária. Diante disso, a criopreservação de células-tronco mesenquimais provenientes de tecido adiposo (CTDAs) poderia ser uma maneira de realizar a coleta em um tempo cirúrgico e após realizar a lipoenxertia de forma fracionada. O dimetilsulfóxido (DMSO) é um criopreservante utilizado em pesquisas com células, porém é potencialmente tóxico, o que impossibilitaria a utilização de CTDAs criopreservadas na prática clínica. Novos criopreservantes celulares, sem toxicidade, vêm sendo descritos na literatura científica experimental, como as substâncias L-prolina e trealose. Com isso, esse trabalho teve como objetivo avaliar a viabilidade de CTDAs criopreservadas com a combinação de L-prolina e trealose, em um período de até 90 dias. Método: Estudo experimental, no qual foram obtidas amostras de lipoaspirado provenientes de 9 pacientes. A fração celular foi processada e congelada com L-prolina (1,5M) + trealose (0,2M), ou com DMSO + soro fetal bovino (SFB), como controle. Após 30 e 90 dias, as amostras foram descongeladas e a viabilidade celular foi avaliada pela técnica de MTT. Resultados: A análise das CTDAs, após 1 e 3 meses de congelamento, indicou que as amostras tratadas com L-prolina + trealose apresentaram viabilidade semelhante àquelas preservadas com DMSO e SFB (p=0,444). Conclusão: A associação de L-prolina e trealose manteve CTDA viáveis por 30 e 90 dias de congelamento, podendo ser uma alternativa como criopreservante celular sem toxicidade e viabilizando o uso de lipoenxertia seriada.

Introduction: Fat grafting is an autologous graft of cells from subcutaneous tissue, which can be used as a complementary technique in breast reconstruction. Given this, the cryopreservation of adipose tissue-derived mesenchymal stem cells (ADMSCs) could be a way to collect them in one surgical procedure and after performing fractional fat grafting. Dimethyl sulfoxide (DMSO) is a cryopreservative used in cell research, but it is potentially toxic, which would make it impossible to use cryopreserved ADMSCs in clinical practice. New cellular cryopreservatives, without toxicity, have been described in the experimental scientific literature, such as the substances L-proline and trehalose. Therefore, this work aimed to evaluate the viability of ADMSCs cryopreserved with the combination of L-proline and trehalose over up to 90 days. Method: Experimental study in which lipoaspirate samples were obtained from 9 patients. The cellular fraction was processed and frozen with L-proline (1.5M) + trehalose (0.2M) or with DMSO + fetal bovine serum (FBS) as control. After 30 and 90 days, the samples were thawed, and cell viability was assessed using the MTT technique. Results: The analysis of ADMSCs, after 1 and 3 months of freezing, indicated that samples treated with L-proline + trehalose showed similar viability to those preserved with DMSO and SFB (p=0.444). Conclusion: The association of L-proline and trehalose kept ADMSC viable for 30 and 90 days of freezing, and could be an alternative as a cellular cryopreservative without toxicity and enabling the use of serial fat grafting.

Aqueous extract of the bark of Uncaria tomentosa, an amazonian medicinal plant, promotes gastroprotection and accelerates gastric healing in rats.

ETHNOPHARMACOLOGICAL IMPORTANCE: Uncaria tomentosa Willd. DC., is used in the Amazonian region of South America, wherein ethnic groups use the plant to treat diseases, including gastric disorders. However, despite its widespread popular use, this species has yet to be assessed for its anti-ulcer effects. AIM OF THE STUDY: In this study, we aimed to evaluate the in vivo gastroprotective and gastric healing activities of an aqueous extract of the bark of Uncaria tomentosa (AEUt) and sought to gain an understanding of the pharmacological mechanisms underlying these biological effects. MATERIALS AND METHODS: To verify the gastroprotective properties rats were treated with AEUt (30, 60, or 120 mg/kg) prior to inducing gastric ulceration with ethanol or piroxicam. Additionally, the involvement of nitric oxide, non-protein sulfhydryl compounds (NP-SH), α-2 adrenergic receptors, and prostaglandins was investigated. Furthermore, a pylorus ligature model was employed to investigate the antisecretory activity of AEUt. The gastric healing effects of AEUt (60 mg/kg) were examined in rats in which ulceration had been induced with 80% acetic acid, whereas the quality of healing was evaluated in mice with interleukin-induced recurrent ulcers. We also evaluated the in vivo thickness of the gastric wall using ultrasonography. Moreover, the levels of reduced glutathione (GSH) and malondialdehyde (MDA) were evaluated in ulcerated mucosa, and we determined the activities of the enzymes myeloperoxidase (MPO), N-acetyl-ß-D-glycosaminidase, superoxide dismutase, catalase, and glutathione S-transferase. In addition, we assessed the effects of AEUt on cell viability and subjected the AEUt to phytochemical analyses. RESULTS: Administration of the AEUt (60 or 120 mg/kg) prevented ethanol- and piroxicam-induced ulceration, which was also confirmed histologically. Moreover, we observed that pre-treatment with NEM and indomethacin abolished the gastroprotective effects of AEUt, thereby indicating the involvement of NP-SH and prostaglandins in these protective effects. In addition, we found that the administration of AEUt had no appreciable effects on the volume, acidity, or peptic activity of gastric juice. Furthermore, the AEUt (60 mg/kg) accelerated the gastric healing of acetic acid-induced ulcers by 46.2% and ultrasonographic findings revealed a reduction in the gastric wall thickness in this group. The gastric healing effect of AEUt was also accompanied by a reduction in MPO activity. The AEUt (60 mg/kg) also minimized ulcer recurrence in mice exposed to IL-1ß and was associated with the maintenance of GSH levels and a reduction in MDA contents. We deduce that the biological effects of AEUt could be associated with the activities of polyphenols and the alkaloids isomitraphylline and mitraphylline, identified as predominant constituents of the AEUt. Furthermore, we found no evidence to indicate that AEUt would have any cytotoxic effects. CONCLUSION: Collectively, our findings provide compelling evidence indicating the therapeutic efficacy of U. tomentosa. Our data indicate that compounds in AEUt confer gastroprotection and that this preventive effect of AEUt was accompanied by gastric healing and a reduction in gastric ulcer recurrence. Moreover, we provide evidence to indicate that the gastroprotective and gastric healing effects involve the antioxidant system and anti-inflammatory responses that contribute to preserving the gastric mucosa.

Resistance training reduces platelet activation in hypertensive women: the role of purinergic signaling.

BACKGROUND AND AIM: Essential arterial hypertension is a risk factor for stroke, myocardial infarction, heart failure, and arterial aneurysm, which are related to the activation of platelets. Purinergic signaling has a central role in platelet aggregation. Although ATP and ADP can act as a proaggregant agent, adenosine inhibits platelet aggregation and reduces vascular injury. Physical exercise exhibits antiaggregant properties and can modulate purinergic system. The aim of this study was to evaluate the effect of 6 months of resistance training on purinergic system components in platelets and on platelet activation, hemodynamic and anthropometric parameters in hypertensive woman. METHOD: A total of 31 hypertensive and 28 normotensive middle-aged sedentary women were submitted to 6 months of resistance training. Purinergic enzymes activities were assessed in platelets; ATP and Tromboxane B2 (TXB2) levels were measured in serum. Blood pressure (BP), BMI, and body fat were also measured. All variables were statistically analyzed, considering P value less than 0.05. RESULTS: Six months of resistance training was able to significantly reduce BP, ATP, and TXB2 levels as well as NTPDase, ecto-5'nucleotidase, and ADA activities in hypertensive group. After 6 months of resistance training, purinergic system components and TXB2 of hypertensive group were similar to normotensive group in platelets, demonstrating that resistance training was able to modulate platelet activation. A positive correlation was found between BP, enzyme activities, and levels of ATP and TXB2. CONCLUSION: Our findings demonstrated the relationship between purinergic signaling and platelet activation in hypertension and suggests that resistance training serve as tool to reduce platelet aggregation in hypertensive woman by modulating purinergic system.

Biochemical analysis of oxidative stress and purinergic system in pregnant women with gestational diabetes mellitus / Análise bioquímica do estresse oxidativo e do sistema purinérgico em gestantes com diabetes mellitus gestacional

Background: Pregnancy is characterized as a physiological period with greater sensitivity to insulin resistance and changes in oxidative stress. Purinergic signaling is directly related to diabetes, as this condition modifies the concentration of extracellular ATP and the level of degradation of ATP to adenosine. Objective: Analyze oxidative stress and the purinergic system in pregnant women with Gestational Diabetes Mellitus (GDM) and compare them with low-risk pregnant women (LR). Materials and Methods: The research was of a quantitative approach of an experimental nature. The study was carried out at the Clínica da Mulher, which serves high-risk pregnant women, and at the Family Health Centers, which serves low-risk pregnant women, both located in Chapecó, Santa Catarina, Brazil. Results: From the analysis, it was observed that oxidative stress was increased in pregnant women in LR compared to pregnant women with GDM by increasing the concentration of TBARS and reducing the concentration of Carbonyl Protein in pregnant women with LR. Regarding the purinergic system, there was a significant decrease in the hydrolysis of the nucleotides ATP, ADP, and AMP in pregnant women with GDM, and a significant increase in the hydrolysis of ADA, also in pregnant women with GDM. Conclusion: Therefore, pregnant women with GDM have less oxidative stress compared to pregnant women in LR concerning TBARS and Carbonyl Protein markers, thus allowing a greater antioxidant defense mechanism. Furthermore, concerning the purinergic system, there is an increase in the activity of ADA, which is directly related to the immunosuppression process, a necessary condition for the protection of the fetus during the gestational period (AU).

Introdução: A gravidez é caracterizada como um período fisiológico em que há uma maior sensibilidade a resistência à insulina e alterações no estresse oxidativo. A sinalização purinérgica está diretamente relacionada ao diabetes, pois esta condição modifica a concentração de ATP extracelular e o nível de degradação de ATP em adenosina. Objetivo:Analisar o estresse oxidativo e o sistema purinérgico em gestantes com Diabetes Mellitus Gestacional (DMG) e compará-los com gestantes de baixo risco (BR). Materiais e Métodos: A pesquisa foi de abordagem quantitativa, de caráter experimental. O estudo foi realizado na Clínica da Mulher, que atende gestantes de alto risco, e nas Unidades de Saúde da Família, que atendem gestantes de baixo risco, ambas localizadas no município de Chapecó, Santa Catarina, Brasil. Resultados: A partir das análises, observou-se que o estresse oxidativo apresentou-se aumentado em gestantes de BR quando comparado a gestantes com DMG. No que tange ao sistema purinérgico, houve uma diminuição significativa na hidrólise dos nucleotídeos ATP, ADP e AMP em gestantes com DMG, bem como um aumento significativo na hidrólise de ADA, também em gestantes com DMG. Conclusão: Portanto, gestantes com DMG possuem menor estresse oxidativo quando comparado a gestantes de BR, permitindo assim, um maior mecanismo de defesa antioxidante. Para mais, no que se refere ao sistema purinérgico, verifica-se o aumento da concentração de ADA está diretamente relacionada ao processo de imunossupressão, condição necessária à proteção do feto durante o período gestacional (AU).

Quetiapine effect on depressive-like behaviors, oxidative balance, and inflammation in serum of rats submitted to chronic stress.

Major depressive disorder (MDD) etiology is still not completely understood, and many individuals resist the traditional treatments. Chronic exposure to stressful events can contribute to development and progression and be involved in biological changes underlying MDD. Among the biological mechanisms involved, inflammatory changes and oxidative balance are associated with MDD pathophysiology. Quetiapine, a second-generation antipsychotic, induces a better therapeutic response in individuals refractory to traditional treatments. The main objectives of this research were as follows: to evaluate the effect of chronic mild stress (CMS) on depressive-like behaviors, oxidative stress, and inflammation in adult rats; to evaluate the possible antidepressant, antioxidant, and anti-inflammatory effects of quetiapine. The animals were submitted to CMS protocols. At the end of the CMS, the animals were submitted to a chronic treatment for 14 days with the following drugs: quetiapine (20 mg/kg), imipramine (30 mg/kg), and escitalopram (10 mg/kg). At the end of the treatments, the animals were evaluated in the open field tests, anhedonia (splash test), and forced swimming. The animals were euthanized after the behavioral tests, and serum samples were collected. Myeloperoxidase (MPO) activity and interleukin-6 (IL-6) levels were analyzed. CMS induced an increase in depressive-like behaviors, and quetiapine significantly reduced these behaviors. MPO activity and IL-6 levels increased in the serum of animals submitted to CMS. Quetiapine significantly reduced MPO activity and IL-6 levels. These results corroborate other evidence, indicating that chronic stress is a relevant phenomenon in the etiology of depression and suggesting that quetiapine induces an antidepressant effect because it reduces oxidative and inflammatory mechanisms.

The anti-inflammatory effect of resistance training in hypertensive women: the role of purinergic signaling.

BACKGROUND AND METHODS: Essential arterial hypertension triggers a chronic inflammatory process that seems to be linked to purinergic signaling. Physical exercise exhibit anti-inflammatory properties and is able to modulates purinergic system. The aim of this study was to evaluate the effect of 6 months of resistance training on inflammatory markers, purinergic system components, hemodynamic and anthropometric parameters in hypertensive woman. METHODS: A total of 31 hypertensive group and 28 normotensive (control group) middle-aged sedentary women were submitted to 6 months of resistance training. All measurements and blood collection were carried out before (pretest), after 3 months and after 6 months (posttest) of training. Purinergic enzymes [nucleoside triphosphate diphosphohydrolase (NTPDase) and adenosine deaminase] were assessed in lymphocytes; IL-6, IL-10, ATP and C-reactive protein levels were measured in serum. RESULTS: Six months of resistance training was able to significantly reduce blood pressure (BP), IL-6, C-reactive protein, ATP levels as well as NTPDase and adenosine deaminase activities in hypertensive group. Physical training was also able to increase IL-10 levels in hypertensive group. A positive correlation was found between BP, enzyme activities and levels of ATP and IL-6. A negative correlation was found between BP and IL-10. Positive correlation was found between NTPDase and IL-6 levels (P < 0.05) as well as ATP levels and IL-6 levels. CONCLUSION: Our findings demonstrated the relationship between purinergic signaling and inflammation in hypertension and suggests that resistance training serve as tool to reduce inflammation in hypertensive woman by modulating purinergic system.

Exercise with blood flow restriction as a new tool for health improvement in hypertensive elderly women: the role of purinergic enzymes.

BACKGROUND: Purinergic signaling has been considered one of the mechanisms by which exercise exerts its antihypertensive effects; and research on the effects of blow flow restriction (BFR) exercise has increased as an alternative for elderly hypertensive patients. We analyzed the acute responses of NTPDase and adenosine deaminase (ADA) activities to low intensity aerobic exercise (LIAE) with BFR in lymphocytes of hypertensive elderly women. METHODS: Sixteen hypertensive elderly women performed three exercise protocols: LIAE; high intensity aerobic exercise (HIAE) and LIAE+BFR. Blood pressure, heart rate and blood collection were carried out before exercise, immediately after exercise and 30 min after exercise. NTPDase and ADA activities were measured in lymphocytes. RESULTS: Our results showed that LIAE+BFR triggered the same stimuli when compared to HIAE exercise regarding to NTPDases activities, suggesting that both protocols trigger an augment of these enzyme activities in response to: 1) increase in ATP release during exercise; and 2) need of adenosine generation to promotes anti-inflammatory responses in the recovery period. HIAE protocol was more effective than the others to trigger combined hypotensive and anti-inflammatory effects in the recovery period. CONCLUSIONS: This study showed that BFR is a good tool to promote anti-inflammatory effects similar (not equal) to HIAE. Moreover, LIAE+BFR promotes much more stimuli and adaptations related to immune functions than low intensity protocols, bringing more benefits for the hypertensive elderly population.

The signaling effects of ATP on melanoma-like skin cancer.

Melanoma is a type of skin cancer originated by the malignant transformation of melanocytes. Increasing incidence and mortality require efforts focused on studies and research about this cancer. Its microenvironment is rich in extracellular ATP, but there are no studies evaluating the ectonucleotidases and ATP effects on tumor-derived melanoma cells with known amounts of ATP. This way, the objective of this work was to evaluate the purinergic signaling in the pathophysiology of in vivo melanoma and the in vitro effects of ATP signaling. We found increased and effective extracellular ATP hydrolysis in platelets and a significant decrease of extracellular ATP levels and adenosine hydrolysis. In addition, we cultured PBMCs of melanoma patients and used ATP salt with specific concentrations to evaluate its signaling effects. The enzymatic activity analysis revealed that even with higher ATP doses cells metabolize adenine nucleotides less efficiently, and present low ATP, ADP and AMP hydrolytic activity in CM compared to CT cells. In summary, we showed for the first time important data about the purinergic signaling in the pathophysiology of melanoma and ATP signaling exercising immunosuppressive effects. Therefore, as already shown for other tumors, the purinergic signaling should be considered a potential target for melanoma management and treatment and could offer novel therapeutic prospects.

Antiproliferative and apoptotic effects of caffeic acid on SK-Mel-28 human melanoma cancer cells.

Cutaneous melanoma (CM) is an extremely aggressive cancer presenting low survival and high mortality. The vast majority of patients affected by this disease does not respond or show resistance to the chemotherapeutic drugs, which makes the treatment ineffective. In this sense, the necessity for the development of new agents to assist in CM therapy is extremely important. One of the sources of great interest in this search are compounds of natural origin. Among these compounds, caffeic acid has demonstrated a broad spectrum of pharmacological activities as well as antitumor effects in some types of cancer. Therefore, the objective of this work was to investigate the possible antitumor effect of caffeic acid on the SK-Mel-28 cell line, human CM cells. Cells were cultured in flasks with culture medium containing fetal bovine serum, antibiotic, and antifungal, and maintained in ideal conditions. Cells were treated with 25 µM, 50 µM, 100 µM, 150 µM and 200 µM of caffeic acid and dacarbazine at 1 mg/mL. We verified the effect on cell viability and cell death, apoptosis, cell cycle, colony formation and gene expression of caspases. Results showed a decrease in cell viability, cell death induction by apoptosis, inhibition of colony formation, modulation of cell cycle and alterations in gene expression of caspases after caffeic acid treatment. These results suggest an antitumor effect of the compound on SK-Mel-28 cells. This study provides original information on mechanisms by which caffeic acid may play a key role in preventing tumor progression in human melanoma cells.

High-intensity intermittent exercise increases adenosine hydrolysis in platelets and lymphocytes and promotes platelet aggregation in futsal athletes.

Acute bouts of high-intensity intermittent exercise (HIIE) or sports are associated with changes in lymphocytes and platelet functions and we hypothesized that the purinergic system is involved with these alterations. We investigated the activity of ectonucleotidases in platelets and lymphocytes as well as the platelet aggregation of futsal players in response to an acute protocol of HIIE. Thus, 19 male semi-professional futsal players were submitted to 40 min of HIIE on a treadmill. Blood samples were collected three-time points: before exercise, immediately after, and 30 min after the end of the session. Platelet-rich plasma (PRP) and lymphocytes were isolated. ATP, ADP, AMP, and adenosine hydrolysis, NTPDase1 (CD39) expression as well as platelet aggregation were measured. Our results showed HIIE induced a decrease in ATP and ADP hydrolysis in platelets, an increase in adenosine hydrolysis and an increase in platelet aggregation immediately after exercise. After 30 min of recovery, enzymatic activity and platelet aggregation returned to baseline levels. In lymphocytes, adenosine hydrolysis was augmented immediately after exercise and remained increased even after 30 min of recovery. In conclusion, acute HIIE triggers a transient proaggregant status that is reverted after a 30 min of recovery. The effects of HIIE in lymphocytes remained after 30 min of recovery, indicating a pro-inflammatory response. This work elucidated some of the mechanisms by which purinergic system regulates lymphocytes and platelets activities related to HIIE, suggesting that the type of exercise may influence an increase in platelet aggregation even in trained individuals.

The Impact of Purinergic System Enzymes on Noncommunicable, Neurological, and Degenerative Diseases.

Evidences show that purinergic signaling is involved in processes associated with health and disease, including noncommunicable, neurological, and degenerative diseases. These diseases strike from children to elderly and are generally characterized by progressive deterioration of cells, eventually leading to tissue or organ degeneration. These pathological conditions can be associated with disturbance in the signaling mediated by nucleotides and nucleosides of adenine, in expression or activity of extracellular ectonucleotidases and in activation of P2X and P2Y receptors. Among the best known of these diseases are atherosclerosis, hypertension, cancer, epilepsy, Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). The currently available treatments present limited effectiveness and are mostly palliative. This review aims to present the role of purinergic signaling highlighting the ectonucleotidases E-NTPDase, E-NPP, E-5'-nucleotidase, and adenosine deaminase in noncommunicable, neurological, and degenerative diseases associated with the cardiovascular and central nervous systems and cancer. In conclusion, changes in the activity of ectonucleotidases were verified in all reviewed diseases. Although the role of ectonucleotidases still remains to be further investigated, evidences reviewed here can contribute to a better understanding of the molecular mechanisms of highly complex diseases, which majorly impact on patients' quality of life.

High levels of extracellular ATP lead to chronic inflammatory response in melanoma patients.

Skin cancer represents a serious public health problem and melanoma is considered the most significant due to its high metastasis capacity. Evasion mechanisms are the main characteristic of these tumor cells to escape of immune response. Extracellular nucleotides and nucleosides play an important role in inflammatory and immune responses. In this study, we analyzed the expression and activity of purinergic system enzymes in platelets and lymphocytes, ATP levels quantification, as well the level of pro and anti-inflammatory interleukins in the serum of 23 patients with surgical melanoma removal (CM group) and 23 control subjects (CT group). Results showed a decrease in ATP, ADP, and AMP hydrolysis and an increase in ATP levels quantification in CM group. The pro-inflammatory cytokines were elevated in CM group when compared to CT group. These results suggest an inflammatory process, even after surgical removal, due to elevated extracellular ATP levels. Besides, CM group displayed an increase in IL-10 levels and an increased in ADA activity in platelets and lymphocytes. Once adenosine and IL-10 are anti-inflammatory molecules, these results indicate a down-regulation of immune system front to malignant process. The alteration in nucleotide and nucleoside hydrolysis reinforces the purinergic systems role in this cancer. Therefore, even after surgical removal, the purinergic system can develop a chronic inflammatory micro-environment that can influence directly on relapse or metastasis.

Relação entre o desenvolvimento do melanoma cutâneo e o estresse oxidativo / Relationship between the development of cutaneous melanoma and oxidative stress

O carcinoma cutâneo é a neoplasia maligna mais frequente na população brasileira, correspondendo a cerca de 25% de todas as lesões malignas registradas no País. Embora represente apenas cerca de 5% dos casos de câncer de pele, o melanoma provoca a maioria das mortes por malignidades cutâneas, pelo seu alto potencial de enviar metástases a órgãos distantes. Os fatores de risco para o desenvolvimento do câncer de pele tanto podem ser genéticos quanto ambientais. O prognóstico desse tipo de câncer pode ser considerado bom se detectado nos estágios iniciais.

The skin carcinoma is the most common malignancy in the Brazilianpopulation, accounting for about 25% of all malignant lesions recorded in Brazil. While representing only about 5% of cases skin cancer, melanoma causes the most deaths from cutaneous malignancies, for its high potential to send metastases to distant organs. Risk factors for the development of skin cancer can be both genetic and environmental. The prognosis of this type of cancer can be considered good if detected in the early stages.