RESUMO
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is upregulated in congestive heart failure (CHF), contributing to electrical, structural, and functional remodeling. CaMKII inhibition is known to improve CHF, but its direct cardiac effects in CHF remain unclear. We hypothesized that CaMKII inhibition improves cardiomyocyte function, [Ca2+]i regulation, and ß-adrenergic reserve, thus improving advanced CHF. In a 16-week study, we compared plasma neurohormonal levels and left ventricular (LV)- and myocyte-functional and calcium transient ([Ca2+]iT) responses in male Sprague-Dawley rats (10/group) with CHF induced by isoproterenol (170 mg/kg sq for 2 days). In rats with CHF, we studied the effects of the CaMKII inhibitor KN-93 or its inactive analog KN-92 (n = 4) (70 µg/kg per day, mini-pump) for 4 weeks. Compared with controls, isoproterenol-treated rats had severe CHF with 5-fold-increased plasma norepinephrine and about 50% decreases in ejection fraction (EF) and LV contractility [slope of LV end-systolic pressure-LV end-systolic volume relation (EES)] but increased time constant of LV relaxation (τ). They also showed significantly reduced myocyte contraction [maximum rate of myocyte shortening (dL/dtmax)], relaxation (dL/dtmax), and [Ca2+]iT Isoproterenol superfusion caused significantly fewer increases in dL/dtmax and [Ca2+]iT KN-93 treatment prevented plasma norepinephrine elevation, with increased basal and acute isoproterenol-stimulated increases in EF and EES and decreased τ in CHF. KN-93 treatment preserved normal myocyte contraction, relaxation, [Ca2+]iT, and ß-adrenergic reserve, whereas KN-92 treatment failed to improve LV and myocyte function, and plasma norepinephrine remained high in CHF. Thus, chronic CaMKII inhibition prevented CHF-induced activation of the sympathetic nervous system, restoring normal LV and cardiomyocyte basal and ß-adrenergic-stimulated contraction, relaxation, and [Ca2+]iT, thereby playing a rescue role in advanced CHF. SIGNIFICANCE STATEMENT: We investigated the therapeutic efficacy of late initiation of chronic Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibition on progression of advanced congestive heart failure (CHF). Chronic CaMKII inhibition prevented CHF-induced activation of the sympathetic nervous system and restored normal intrinsic cardiomyocyte basal and ß-adrenergic receptor-stimulated relaxation, contraction, and [Ca2+]i regulation, leading to reversal of CHF progression. These data provide new evidence that CaMKII inhibition is able and sufficient to rescue a failing heart, and thus cardiac CaMKII inhibition is a promising target for improving CHF treatment.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Animais , Insuficiência Cardíaca , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To better understand the contribution of age to the development of osteoarthritis (OA). METHODS: Surgical destabilization of the medial meniscus (DMM) was used to model OA in 12-week-old and 12-month-old male C57BL/6 mice. OA severity was evaluated histologically. RNA used for microarray and real-time polymerase chain reaction analysis was isolated from joint tissue collected from the medial side of the joint, including cartilage, meniscus, subchondral bone, and the joint capsule with synovium. Computational analysis was used to identify patterns of gene expression, and immunohistochemistry was used to evaluate tissue distribution of selected proteins. RESULTS: OA was more severe in older mice than in young mice. Only 55 genes showed a similar expression with DMM-induced OA in the 2 age groups, while 493 genes showed differential expression, the majority having increased expression in older mice. Functional categories for similarly expressed genes included extracellular matrix- and cell adhesion-related genes; differentially expressed genes included those related to muscle structure and development and immune response genes. Comparison of expression in sham-operated control joints revealed an age-related decrease in matrix gene expression and an increase in immune and defense response gene expression. Interleukin-33 was present in multiple joint tissue cells, while CCL21 was more localized to chondrocytes and meniscal cells. Periostin was found in the extracellular matrix of cartilage and meniscus. CONCLUSION: Age affects both the basal pattern of gene expression in joint tissues and the response to surgically induced OA. Examining tissue from the joint beyond only cartilage revealed novel genes and proteins that would be important to consider in OA.
Assuntos
Fatores Etários , Artrite Experimental/genética , Regulação da Expressão Gênica , Osteoartrite/genética , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Quimiocina CCL21/genética , Quimiocina CCL21/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Membro Posterior , Interleucina-33 , Interleucinas/genética , Interleucinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Osteoartrite/metabolismo , Osteoartrite/patologia , Joelho de Quadrúpedes/metabolismo , Joelho de Quadrúpedes/patologia , Joelho de Quadrúpedes/cirurgia , Tíbia/cirurgiaRESUMO
BACKGROUND: The melanocortin 4 antagonist TCMCB07 is safe and effective in reversing cachexia caused by sepsis or cancer in rodents. The safety and pharmacokinetics of TCMCB07 are demonstrated in healthy beagle dogs. HYPOTHESIS/OBJECTIVES: The objectives of this study were to investigate the safety, peak plasma concentrations, and potential for efficacy of TCMCB07 in pet dogs with naturally occurring cachexia over a 4-week time period. ANIMALS: Fourteen dogs with cachexia of any underlying cause, except cancer of the oral cavity or gastrointestinal tract, were eligible for enrollment with informed client consent. METHODS: This study was a prospective, 1-armed open-label trial. Physical examination, complete blood count, chemistry panel, and owner-assessed quality of life surveys were checked at weeks 1, 2, and 4. Due to potential for bradycardia and hypotension, Holter monitoring and blood pressure evaluations were scheduled at pre-enrollment and week 4. RESULTS: Fourteen dogs completed the trial. Significant changes detected included increased mean body weight (18.6-19.5 kg, P < .02), increased body condition score (median Tufts 5-point thin dog scale score P < .004 and WSAVA muscle condition score P < .02) and increased mean blood urea nitrogen (21.79-30.43 mg dL-1 , P < .004). On quality of life surveys, pet owners perceived their dog appeared to be panting less (P < .002) and that the general health improved (P < .03). Four dogs had a change in coat pigmentation. The peak plasma concentration of TCMCB07 in cachectic dogs was similar to that in healthy beagle dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: TCMCB07 was safe and has potential efficacy in pet dogs with cachexia.
Assuntos
Doenças do Cão , Neoplasias , Humanos , Animais , Cães , Caquexia/tratamento farmacológico , Caquexia/veterinária , Estudos Prospectivos , Qualidade de Vida , Melanocortinas , Peptídeos , Neoplasias/veterinária , Doenças do Cão/tratamento farmacológicoRESUMO
Hydroxyproline (Hyp) metabolism is a key source of glyoxylate production in the body and may be a major contributor to excessive oxalate production in the primary hyperoxalurias where glyoxylate metabolism is impaired. Important gaps in our knowledge include identification of the tissues with the capacity to degrade Hyp and the development of model systems to study this metabolism and how to suppress it. The expression of mRNA for enzymes in the pathway was examined in 15 different human tissues. Expression of the complete pathway was identified in liver, kidney, pancreas, and small intestine. HepG2 cells also expressed these mRNAs and enzymes and were shown to metabolize Hyp in the culture medium to glycolate, glycine, and oxalate. [(18)O]- and [(13)C(5)]Hyp were synthesized and evaluated for their use with in vitro and in vivo models. [(18)O]Hyp was not suitable because of an apparent tautomerism of [(18)O]glyoxylate between enol and hydrated forms, which resulted in a loss of isotope. [(13)C(5)]Hyp, however, was metabolized to [(13)C(2)]glycolate, [(13)C(2)]glycine, and [(13)C(2)]oxalate in vitro in HepG2 cells and in vivo in mice infused with [(13)C(5)]Hyp. These model systems should be valuable tools for exploring various aspects of Hyp metabolism and will be useful in determining whether blocking Hyp catabolism is an effective therapy in the treatment of primary hyperoxaluria.
Assuntos
Hidroxiprolina/metabolismo , Hiperoxalúria/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Isótopos de Carbono , Regulação da Expressão Gênica/fisiologia , Células Hep G2 , Humanos , Rim/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estrutura Molecular , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
PURPOSE: Botulinum neurotoxin-A (BoNTA) is used to treat several disorders, including Raynaud phenomenon. Recent investigations cite toxin-induced increases in blood flow, but no mechanism for BoNTA's actions is proposed. This study hypothesized that local application of BoNTA causes arteriolar vasodilation through sympathetic blockade and results in increased blood flow. METHODS: Microvascular effects of BoNTA were assessed using a rat cremaster preparation. Cremaster microvascular diameters were measured in the muscle before and after treatment with the muscle paralytic agent gallamine triethiodide. Preparations were then treated with one of the following: BoNTA (4, 6, or 10 units), BoNTA dilution vehicle, or denatured BoNTA. Arteriolar diameters were measured repeatedly over the observation period. Additional preparations were treated with either tetrodotoxin or prazosin and rauwolscine before BoNTA to confirm that the observed vasodilatory responses were the result of sympathetic neural inhibition. RESULTS: The BoNTA application resulted in a significant dose-dependent vasodilation (13% to 15%) of observed cremaster arterioles. Control treatments did not cause vasodilation. Both tetrodotoxin and prazosin/rauwolscine treatments elicited similar vasodilatory effects, with no additional vasodilation elicited by BoNTA. Addition of sodium nitroprusside following BoNTA elicited further vasodilation. In addition, systemic arterial pressure was unaffected by the local administration of BoNTA. CONCLUSIONS: Local application of BoNTA results in arteriolar dilation that yields an approximate 69% increase in blood flow, without changing systemic arterial pressure. A BoNTA-mediated vasodilation through sympathetic blockade is a likely mechanism to explain the increase in blood flow reported after treatment with the toxin. CLINICAL RELEVANCE: The ability of BoNTA to inhibit sympathetic nervous input reduces vasoconstriction, which is the most likely mechanism for improvement seen in Raynaud phenomenon patients following BoNTA injection.
Assuntos
Toxinas Botulínicas Tipo A/farmacologia , Fármacos Neuromusculares/farmacologia , Antagonistas de Receptores Adrenérgicos alfa 2/farmacologia , Animais , Relação Dose-Resposta a Droga , Humanos , Masculino , Microcirculação/efeitos dos fármacos , Prazosina/farmacologia , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional/efeitos dos fármacos , Tetrodotoxina/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Ioimbina/farmacologiaRESUMO
PURPOSE: The chemical denervation that results from botulinum neurotoxin A (BoNT-A) causes a temporary, reversible paresis that can result in easier surgical manipulation of the muscle-tendon unit in the context of tendon rupture and repair. The purpose of the study was to determine whether BoNT-A injections can be used to temporarily and reversibly modulate active and passive skeletal muscle properties. METHODS: Male CD1 mice weighing 40-50 g were divided into a 1-week postinjection group (n = 13: n = 5 saline and n = 8 BoNT-A) and a 2-week postinjection group (n = 17: n = 7 saline and n = 10 BoNT-A). The animals had in vivo muscle force testing and in vivo biomechanical evaluation. RESULTS: There was a substantial decline in the maximal single twitch amplitude (p < .05) and tetanic amplitude (p < .05) at one week and at 2 weeks after BoNT-A injection, when compared to saline-injected controls. BoNT-A injection significantly reduced the peak passive properties of the muscle-tendon unit as a function of displacement at one week (p < .05). Specifically, the stiffness of the BoNT-A injected muscle-tendon unit was 0.417 N/mm compared to the control saline injected group, which was 0.634 N/mm, a 35% reduction in stiffness (p < .05). CONCLUSIONS: Presurgical treatment with BoNT-A might improve the surgical manipulation of the muscle-tendon unit, thus improving surgical outcomes. The results implicate neural tone as a substantial contributor to the passive repair tension of the muscle-tendon unit. The modulation of neural tone through temporary, reversible paresis is a novel approach that might improve intraoperative and postoperative passive muscle properties, allowing for progressive rehabilitation while protecting the surgical repair site.
Assuntos
Toxinas Botulínicas Tipo A/administração & dosagem , Denervação Muscular/métodos , Animais , Modelos Animais de Doenças , Injeções Intramusculares , Masculino , Camundongos , Camundongos Endogâmicos , Força Muscular/efeitos dos fármacos , Músculo Esquelético/cirurgia , Procedimentos Ortopédicos/métodos , Cuidados Pré-Operatórios/métodos , Probabilidade , Distribuição Aleatória , Valores de Referência , Sensibilidade e Especificidade , Estatísticas não Paramétricas , Tendões/cirurgiaRESUMO
Effects of repeated H-Wave® device stimulation (HWDS) on blood flow and angiogenesis in the rat hind limb were studied. The hypothesis tested was that HWDS acutely increases hind limb blood flow, and that repeated HWDS would elicit angiogenesis. Animals were HWDS-conditioned (``Conditioned'') or sham-stimulated (``Sham'') (n = 5/group) daily for 3 weeks. The contralateral limb in both groups served as the control. Each animal was injected with bromodeoxyuridine (BrDU). After 3 weeks, rats were anesthetized and iliac artery blood flow was measured bilaterally before, during, and after acute HWDS. HWDS of the Conditioned limbs elicited a 247% increase in blood flow above resting conditions compared to a 200% increase in control legs. Sham animals did not demonstrate between-leg differences in flow. Hindlimb musculature staining for BrDU revealed angiogenesis in Conditioned versus Sham groups. Flow changes accompanying HWDS corroborated earlier microvascular findings demonstrating a significant striated muscle arteriolar dilation with HWDS.
Assuntos
Terapia por Estimulação Elétrica , Neovascularização Fisiológica , Fluxo Sanguíneo Regional , Animais , Estudos Longitudinais , Extremidade Inferior/irrigação sanguínea , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The melanocortin-4 receptor (MC4R) antagonistic peptide TCMCB07 was developed for the treatment of cachexia. The objectives of this study were to examine pharmacokinetics and safety of TCMCB07 administered subcutaneously to healthy dogs. Dogs were treated with high- (2.25 mg kg-1 ) (n = 5) and low-dose TCMCB07 (0.75 mg kg-1 ) (n = 5) once daily for 28 days with a 14-day washout period between groups. Histamine levels, complete blood count, chemistry panel, blood pressure, 24-hour Holter recording, and pharmacokinetic parameters were monitored in the high-dose group. Physical examination changes were limited to weight gain and darkening of the coat color. There was no elevation of plasma histamine within 24 hours of injection but there was a significant elevation of plasma histamine across time. An approximately doubled eosinophil count and an approximately 25% increase, and then 25% decrease back to pre-treatment plasma phosphorous were also found, although both remained within the reference interval. Serial blood pressure and 24-hour Holter monitors revealed no clinically relevant changes. A difference was found in the AUC between dosing groups and a significant effect of dose, time, and interaction was noted for Vd . Low-dose TCMCB07 had a Cmax of 2.1 ug ml-1 at day 28, compared to high-dose TCMCB07 which had a Cmax 3.6 ug ml-1 at day 28. Once-daily subcutaneous administration of TCMCB07 was well-tolerated for up to 28 days in dogs when administered at doses one and three times (0.75 mg kg-1 and 2.25 mg kg-1 ) the predicted therapeutic dose and pharmacokinetic parameters are described. SIGNIFICANCE STATEMENT: Melanocortin-4 receptor (MC4R) antagonistic peptide TCMCB07 is safe at both low and high doses in dogs. Therapy was tolerated well as determined by physical examination, clinical pathology, and cardiovascular parameters; darkening of the coat was noted with treatment and resolved with discontinuation. Pharmacokinetics are described and further study in the naturally occurring canine model is warranted.
Assuntos
Peptídeos Cíclicos/farmacocinética , Receptor Tipo 4 de Melanocortina/antagonistas & inibidores , Animais , Arritmias Cardíacas/induzido quimicamente , Pressão Arterial/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Cães , Feminino , Histamina/sangue , Injeções Subcutâneas , Masculino , Peptídeos Cíclicos/efeitos adversos , Fósforo/sangueRESUMO
OBJECTIVE: Meniscus injury and the hypoxia-inducible factor (HIF) pathway are independently linked to osteoarthritis pathogenesis, but the role of the meniscus HIF pathway remains unclear. We sought to identify and evaluate HIF pathway response in normal and osteoarthritic meniscus and to examine the effects of Epas1 (HIF-2α) insufficiency in mice on early osteoarthritis development. METHODS: Normal and osteoarthritic human meniscus specimens were obtained and used for immunohistochemical evaluation and cell culture studies for the HIF pathway. Meniscus cells were treated with pro-inflammatory stimuli, including interleukins (IL)-1ß, IL-6, transforming growth factor (TGF)-α, and fibronectin fragments (FnF). Target genes were also evaluated with HIF-1α and HIF-2α (Epas1) overexpression and knockdown. Wild-type (n = 36) and Epas1+/- (n = 30) heterozygous mice underwent destabilization of the medial meniscus (DMM) surgery and were evaluated at 2 and 4 weeks postoperatively for osteoarthritis development using histology. RESULTS: HIF-1α and HIF-2α immunostaining and gene expression did not differ between normal and osteoarthritic meniscus. While pro-inflammatory stimulation significantly increased both catabolic and anabolic gene expression in the meniscus, HIF-1α and Epas1 expression levels were not significantly altered. Epas1 overexpression significantly increased Col2a1 expression. Both wild-type and Epas1+/- mice developed osteoarthritis following DMM surgery. There were no significant differences between genotypes at either time point. CONCLUSION: The HIF pathway is likely not responsible for osteoarthritic changes in the human meniscus. Additionally, Epas1 insufficiency does not protect against osteoarthritis development in the mouse at early time points after DMM surgery. The HIF pathway may be more important for protection against catabolic stress.
Assuntos
Menisco , Osteoartrite , Animais , Condrócitos/metabolismo , Hipóxia/metabolismo , Hipóxia/patologia , Meniscos Tibiais/patologia , Menisco/metabolismo , Camundongos , Osteoartrite/metabolismoRESUMO
Cachexia, a devastating wasting syndrome characterized by severe weight loss with specific losses of muscle and adipose tissue, is driven by reduced food intake, increased energy expenditure, excess catabolism, and inflammation. Cachexia is associated with poor prognosis and high mortality and frequently occurs in patients with cancer, chronic kidney disease, infection, and many other illnesses. There is no effective treatment for this condition. Hypothalamic melanocortins have a potent and long-lasting inhibitory effect on feeding and anabolism, and pathophysiological processes increase melanocortin signaling tone, leading to anorexia, metabolic changes, and eventual cachexia. We used 3 rat models of anorexia and cachexia (LPS, methylcholanthrene sarcoma, and 5/6 subtotal nephrectomy) to evaluate efficacy of TCMCB07, a synthetic antagonist of the melanocortin-4 receptor. Our data show that peripheral treatment using TCMCB07 with intraperitoneal, subcutaneous, and oral administration increased food intake and body weight and preserved fat mass and lean mass during cachexia and LPS-induced anorexia. Furthermore, administration of TCMCB07 diminished hypothalamic inflammatory gene expression in cancer cachexia. These results suggest that peripheral TCMCB07 treatment effectively inhibits central melanocortin signaling and therefore stimulates appetite and enhances anabolism, indicating that TCMCB07 is a promising drug candidate for treating cachexia.
Assuntos
Caquexia/tratamento farmacológico , Receptor Tipo 4 de Melanocortina/antagonistas & inibidores , Insuficiência Renal Crônica/tratamento farmacológico , Sarcoma Experimental/tratamento farmacológico , Animais , Apetite/efeitos dos fármacos , Caquexia/etiologia , Caquexia/metabolismo , Caquexia/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Receptor Tipo 4 de Melanocortina/metabolismo , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Sarcoma Experimental/complicações , Sarcoma Experimental/metabolismo , Sarcoma Experimental/patologiaRESUMO
Angiotensin-(1-7) [Ang-(1-7)] is an endogenous peptide of the renin-angiotensin system with vasodilator and antiproliferative properties. Our previous studies showed that Ang-(1-7) reduced serum-stimulated growth of human lung cancer cells in vitro through activation of a unique AT((1-7)) receptor. The current study investigates the effect of Ang-(1-7) on lung tumor growth in vivo, using a human lung tumor xenograft model. Athymic mice with tumors resulting from injection of A549 human lung cancer cells were treated for 28 days with either i.v. saline or Ang-(1-7), delivered by implanted osmotic mini-pumps. Treatment with Ang-(1-7) reduced tumor volume by 30% compared with the size before treatment; in contrast, tumor size in the saline-treated animals increased 2.5-fold. These results correlate with a reduction in the proliferation marker Ki67 in the Ang-(1-7)-infused tumors when compared with the saline-infused tumor tissues. Treatment with Ang-(1-7) significantly reduced cyclooxygenase-2 (COX-2) mRNA and protein in tumors of Ang-(1-7)-infused mice when compared with mice treated with saline as well as in the parent A549 human lung cancer cells in tissue culture. These results suggest that Ang-(1-7) may decrease COX-2 activity and proinflammatory prostaglandins to inhibit lung tumor growth. In contrast, the heptapeptide had no effect on COX-1 mRNA in xenograft tumors or A549 cells. Because Ang-(1-7), a peptide with antithrombotic properties, reduces growth through activation of a selective AT((1-7)) receptor, our results suggest that the heptapeptide represents a novel treatment for lung cancer by reducing COX-2.
Assuntos
Adenocarcinoma/tratamento farmacológico , Angiotensina I/farmacologia , Ciclo-Oxigenase 2/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The burden of chronic soft tissue inflammation and neuropathic pain on individuals and society is substantial. This study was conducted to evaluate the H-wave device--an innovative form of treatment for chronic pain and inflammation--in patients with persistent pain associated with injuries or conditions affecting the upper or lower extremities or the back. Patients with at least moderate pain despite conventional therapy were included in a systematic survey after they had been given 2 to 6 wk of treatment with the H-wave device. Measures of improvement involved the proportion of patients with diminished medication requirements, improved function, or pain relief greater than 25%. More than 60% of patients with pain in the lower extremities, upper extremities, or back experienced pain relief exceeding 25%. The proportion of patients whose function improved and who were able to perform a new activity was consistently greater than 50% across the 3 anatomic subgroups. More than 40% of patients in each group were able to reduce or completely eliminate the use of pain medications. These benefits of treatment were independent of the type of pain therapy administered previously. In each anatomic subgroup, the proportion of patients who reported improvement on more than 1 of the 3 endpoints was significantly higher than the expected response to placebo therapy (P<.001). Results suggest that the H-wave device provided important benefits to patients with chronic soft tissue inflammation and neuropathic pain.
Assuntos
Terapia por Estimulação Elétrica/métodos , Inflamação/terapia , Manejo da Dor , Doenças do Sistema Nervoso Periférico/terapia , Doença Crônica , Interpretação Estatística de Dados , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inquéritos e QuestionáriosRESUMO
BACKGROUND: Altered nitric oxide synthase (NOS) has been implicated in the pathophysiology of heart failure (HF). Recent evidence links hypothyroidism to the pathology of HF. However, the precise mechanisms are incompletely understood. The alterations and functional effects of cardiac NOS in hypothyroidism are unknown. We tested the hypothesis that hypothyroidism increases cardiomyocyte inducible NOS (iNOS) expression, which plays an important role in hypothyroidism-induced depression of cardiomyocyte contractile properties, [Ca(2+)]i transient ([Ca(2+)]iT), and ß-adrenergic hyporesponsiveness. METHODS AND RESULTS: We simultaneously evaluated LV functional performance and compared myocyte three NOS, ß-adrenergic receptors (AR) and SERCA2a expressions and assessed cardiomyocyte contractile and [Ca(2+)]iT responses to ß-AR stimulation with and without pretreatment of iNOS inhibitor (1400 W, 10(-5)mol/L) in 26 controls and 26 rats with hypothyroidism induced by methimazole (~30 mg/kg/day for 8 weeks in the drinking water). Compared with controls, in hypothyroidism, total serum T3 and T4 were significantly reduced followed by significantly decreased LV contractility (EES) with increased LV time constant of relaxation. These LV abnormalities were accompanied by concomitant significant decreases in myocyte contraction (dL/dtmax), relaxation (dR/dtmax), and [Ca(2+)]iT. In hypothyroidism, isoproterenol (10(-8)M) produced significantly smaller increases in dL/dtmax, dR/dtmax and [Ca(2+)]iT. These changes were associated with decreased ß1-AR and SERCA2a, but significantly increased iNOS. Moreover, only in hypothyroidism, pretreatment with iNOS inhibitor significantly improved basal and isoproterenol-stimulated myocyte contraction, relaxation and [Ca(2+)]iT. CONCLUSIONS: Hypothyroidism produces intrinsic defects of LV myocyte force-generating capacity and relaxation with ß-AR desensitization. Up-regulation of cardiomyocyte iNOS may promote progressive cardiac dysfunction in hypothyroidism.
Assuntos
Regulação Enzimológica da Expressão Gênica , Hipotireoidismo/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Receptores Adrenérgicos beta/metabolismo , Disfunção Ventricular Esquerda/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Hipotireoidismo/fisiopatologia , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Radiation-induced fibrosis (RIF) of musculoskeletal tissue is a common complication of radiation therapy for extremity soft-tissue sarcoma, with no standardized strategy for prevention and treatment. Angiotensin-(1-7) (Ang-[1-7]), a well-tolerated endogenous heptapeptide hormone with antitumor and antifibrotic properties, was tested as a radioprotectant for RIF and stiffening of irradiated muscles. METHODS: Male CD-1 mice were randomized to one of three treatment groups: control, simulated sarcoma radiation therapy to the gastrocnemius and soleus muscles, or radiation therapy along with continuous Ang-(1-7) delivery initiated three days before radiation therapy. The biologically equivalent dose of radiation (â¼100.3 Gy) absorbed by normal musculature during the course of radiation therapy for extremity sarcoma was delivered by means of four dose fractions of 7.3 Gy over two weeks. Fibrosis (n = 5 per group) and mechanical properties (n = 4 to 6 per group) of the muscles were measured at six weeks and four months after radiation therapy, and the intramuscular concentration of the profibrotic cytokines transforming growth factor-beta (TGF-ß) and connective tissue growth factor (CTGF) (n = 8 to 10 per group) were measured at six weeks. RESULTS: Interstitial (p < 0.01) and perivascular (p < 0.05) fibrosis increased significantly in the muscles treated with radiation therapy alone versus the nonirradiated controls at both six weeks (interstitial, +89%; perivascular, +112%) and four months (interstitial, +154%; perivascular, +88%). The muscles treated with radiation alone also exhibited increased tension (p < 0.01) versus nonirradiated controls at both six weeks (+779%) and four months (+1761%) when placed under 5% strain, and at four months (+1390%; p < 0.001) under 10% strain. At four months, muscle stiffness had increased in the mice treated with radiation therapy alone (+90%; p = 0.002) compared with nonirradiated controls. TGF-ß production was also greater in this group at six weeks (+37%; p = 0.06) versus control. Ang-(1-7) administration prevented RIF and stiffening, with no differences observed for any other outcome between those receiving radiation therapy with Ang-(1-7) and the nonirradiated controls. Likewise, Ang-(1-7) mitigated the increase in TGF-ß and CTGF concentration from radiation therapy. CONCLUSIONS: Ang-(1-7) attenuated RIF, stiffening, and production of profibrotic cytokines that were elevated in mouse skeletal muscles after simulated radiation therapy for extremity sarcoma. CLINICAL RELEVANCE: Ang-(1-7) may serve as a potential therapy for the prevention of RIF in patients who require radiation therapy as adjuvant treatment for soft-tissue sarcoma.
Assuntos
Angiotensina I/administração & dosagem , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/efeitos da radiação , Fragmentos de Peptídeos/administração & dosagem , Sarcoma Experimental/terapia , Espasmo/prevenção & controle , Análise de Variância , Animais , Biópsia por Agulha , Modelos Animais de Doenças , Fibrose/etiologia , Fibrose/prevenção & controle , Membro Posterior , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos , Neoplasias Musculares/patologia , Neoplasias Musculares/terapia , Músculo Esquelético/patologia , Distribuição Aleatória , Valores de Referência , Sarcoma Experimental/patologia , Sensibilidade e Especificidade , Espasmo/patologiaRESUMO
Absorbable collagen sponges (ACS) are used clinically as carriers of recombinant human bone morphogenetic protein 2 (rhBMP-2) to promote bone regeneration. ACS exhibit ectopic bone growth due to delivery of supraphysiological levels of rhBMP-2, which is particularly problematic in craniofacial bone injuries for both functional and esthetic reasons. We hypothesized that hydrogels from the reduced form of keratin proteins (kerateine) would serve as a suitable alternative to ACS carriers of rhBMP-2. The rationale for this hypothesis is that keratin biomaterials degrade slowly in vivo, have modifiable material properties, and have demonstrated capacity to deliver therapeutic agents. We investigated kerateine hydrogels and freeze-dried scaffolds as rhBMP-2 carriers in a critically-sized rat mandibular defect model. ACS, kerateine hydrogels, and kerateine scaffolds loaded with rhBMP-2 achieved bridging in animals by 8 weeks as indicated by micro-computed tomography. Kerateine scaffolds achieved statistically increased bone mineral density compared to ACS and kerateine hydrogels, with levels reaching those of native bone. Importantly, both kerateine hydrogels and kerateine scaffolds had significantly less ectopic bone growth than ACS sponges at both 8 and 16 weeks post-operatively. These studies demonstrate the suitability of keratins as rhBMP-2 carriers due to equal regenerative capacity with reduced ectopic growth compared to ACS.
Assuntos
Materiais Biocompatíveis , Desenvolvimento Ósseo , Proteína Morfogenética Óssea 2/administração & dosagem , Queratinas/química , Mandíbula/anormalidades , Animais , Regeneração Óssea , Masculino , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Reologia , Alicerces TeciduaisRESUMO
Death after severe hemorrhage remains an important cause of mortality in people under 50 years of age. Keratin resuscitation fluid (KRF) is a novel resuscitation solution made from keratin protein that may restore cardiovascular stability. This postulate was tested in rats that were exsanguinated to 40% of their blood volume. Test groups received either low or high volume resuscitation with either KRF or lactated Ringer's solution. KRF low volume was more effective than LR in recovering cardiac function, blood pressure and blood chemistry. Furthermore, in contrast to LR-treated rats, KRF-treated rats exhibited vital signs that resembled normal controls at 1-week.
Assuntos
Coloides/administração & dosagem , Hemodinâmica , Hipovolemia/terapia , Queratinas/administração & dosagem , Ressuscitação/métodos , Animais , Artérias Carótidas/cirurgia , Modelos Animais de Doenças , Humanos , Hipovolemia/cirurgia , Soluções Isotônicas/administração & dosagem , Masculino , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Lactato de Ringer , Estados UnidosRESUMO
OBJECTIVE: To determine whether the ratio of dietary calcium and oxalate consumption at mealtime affects gastrointestinal oxalate absorption and urinary oxalate excretion. METHODS: A study was conducted with 10 non-stone-forming adults placed on controlled diets with daily calcium and oxalate contents of 1000 and 750 mg, respectively. Subjects consumed a balanced calcium/oxalate ratio diet for 1 week, observed a minimum 1-week washout period, and subsequently consumed an imbalanced calcium/oxalate ratio diet for one week. Urine specimens were collected on the last 4 days of each diet. Outcome measures included urinary creatinine, calcium, and oxalate as well as the Tiselius index for assessing urinary calcium oxalate supersaturation. RESULTS: Total daily calcium excretion, oxalate excretion, and Tiselius index were similar between balanced and imbalanced dietary phases. There were significant differences in calcium excretion (mg/g creatinine) between balanced and imbalanced diets in the 1-6 PM (83.1 vs 110.2, P <.04), 6-11 PM (71.3 vs 107.2, P <.02), and 11 PM-8 AM collections (55.0 vs 41.8, P <.02). There was significantly higher oxalate excretion on the balanced diet in the 1-6 pm time period (28.1 vs 16.7, P <.01). There were no differences in the Tiselius index in these collections. CONCLUSION: These results demonstrate that the sequence of ingesting relatively large amounts of oxalate does not significantly affect calcium oxalate stone risk if the recommended daily quantity of dietary calcium is consumed.
Assuntos
Oxalato de Cálcio/metabolismo , Dieta , Cálculos Renais/epidemiologia , Oxalatos/metabolismo , Adulto , Cálcio da Dieta/urina , Creatinina/urina , Feminino , Humanos , Absorção Intestinal , Cálculos Renais/química , Cálculos Renais/metabolismo , Masculino , Oxalatos/administração & dosagem , Oxalatos/urinaRESUMO
Botulinum Neurotoxin A (BoNT-A) injections have been used for the treatment of muscle contractures and spasticity. This study assessed the influence of (BoNT-A) injections on passive biomechanical properties of the muscle-tendon unit. Mousegastrocnemius muscle (GC) was injected with BoNT-A (n = 18) or normal saline (n = 18) and passive, non-destructive, in vivo load relaxation experimentation was performed to examine how the muscle-tendon unit behaves after chemical denervation with BoNT-A. Injection of BoNT-A impaired passive muscle recovery (15% vs. 35% recovery to pre-stretching baseline, p < 0.05) and decreased GC stiffness (0.531 ± 0.061 N/mm vs. 0.780 ± 0.037 N/mm, p < 0.05) compared to saline controls. The successful use of BoNT-A injections as an adjunct to physical therapy may be in part attributed to the disruption of the stretch reflex; thereby modulating in vivo passive muscle properties. However, it is also possible that BoNT-A injection may alter the structure of skeletal muscle; thus modulating the in vivo passive biomechanical properties of the muscle-tendon unit.
Assuntos
Toxinas Botulínicas Tipo A/farmacologia , Músculo Esquelético/efeitos dos fármacos , Fármacos Neuromusculares/farmacologia , Reflexo de Estiramento/efeitos dos fármacos , Tendões/efeitos dos fármacos , Animais , Estimulação Elétrica , Injeções Intramusculares , Masculino , Camundongos , Modelos Animais , Músculo Esquelético/fisiologia , Tendões/fisiologiaRESUMO
Experiments in humans and rodents using oral doses of glycine and phenylalanine have suggested that the metabolism of these amino acids contributes to urinary oxalate excretion. To better define this contribution, we have examined the primed, constant infusion of [1-(13)C(1)] phenylalanine and [1,2-(13)C(2)] glycine in the postabsorptive state in healthy adults. Subjects were infused for 5 hours, hourly urines were collected, and blood was drawn every 30 minutes. Ion chromatography/mass spectrometry was used to measure [(13)C] enrichment in urinary oxalate, glycolate, and hippurate; and the enrichment of (13)C-amino acids in plasma samples was measured by gas chromatography/mass spectrometry. Following infusion with either 6 µmol/(kg h) [1-(13)C(1)] phenylalanine or 6 µmol/(kg h) [1,2-(13)C(2)] glycine, no isotopic glycolate or oxalate was detected in urine. Based on the limits of detection of our ion chromatography/mass spectroscopy method, these data indicate that less than 0.7% of the urinary oxalate could be derived from phenylalanine catabolism and less than 5% from glycine catabolism. Infusions with high levels of [1,2-(13)C(2)] glycine, 60 µmol/(kg h), increased mean plasma glycine by 29% and the whole-body flux of glycine by 72%. Under these conditions, glycine contributed 16.0% ± 1.6% and 16.6% ± 3.2% to urinary oxalate and glycolate excretion, respectively. Experiments using cultured hepatoma cells demonstrated that only at supraphysiological levels (>1 mmol/L) did glycine and phenylalanine metabolism increase oxalate synthesis. These data suggest that glycine and phenylalanine metabolism make only minor contributions to oxalate synthesis and urinary oxalate excretion.
Assuntos
Glicina/metabolismo , Oxalatos/metabolismo , Oxalatos/urina , Fenilalanina/metabolismo , Adulto , Feminino , Glicina/administração & dosagem , Glicina/sangue , Glicolatos/urina , Células Hep G2 , Hipuratos/urina , Humanos , Masculino , Fenilalanina/administração & dosagem , Fenilalanina/sangueRESUMO
Botulinum neurotoxin-A (BoNTA) is a potent neurotoxin used to alter muscle tone to manage spasticity and to provide tendon bioprotection; however, the appropriate dose and injection volume to administer is not defined. Male mice (n = 120) received BoNTA injections into one gastrocnemius with either a constant volume (10 µl) with a variable dose (1, 3, 6 U/kg) or a constant dose (3 U/kg) in a variable volume (2.5, 5, 10, 20, 30 µl). Electromyographic (EMG) examination, muscle force generation (MFG), and wet muscle mass were measured in the ipsilateral and contralateral limbs at 1, 2, 4, or 12 weeks post-injection. MFG and EMG responses decreased to approximately 40% of contralateral after a 1 U/kg injection and 0% of contralateral by 3 and 6 U/kg injection at 1 week after injection. Neuromuscular blockade was greatest with a 10 µl injection volume. MFG, EMG examination, and wet muscle mass reached contralateral values 12 weeks after injection for all injection doses and volumes tested. Effective injection doses and volumes were identified for producing full and partial neuromuscular blockade in the mouse gastrocnemius. These findings have important clinical implications in the intramuscular administration of BoNTA to manage muscle tone.