Measurement Of Collagen Content Research Articles

Influence of anti-sclerostin monoclonal antibody in the repair of post-extraction sockets of ovariectomized rats

ObjectiveThis study assessed the impact of an anti-sclerostin monoclonal antibody (Scl-Ab)-based osteoporosis drug on the post-extraction alveolar repair of ovariectomized rats. DesignFifteen female rats were randomly distributed into three groups: CTR (healthy animals), OST (osteoporosis induced by ovariectomy), and OST+Scl-Ab (osteoporosis induction followed by Scl-Ab treatment). Ovariectomy or sham surgery was performed 30 days before baseline, and Scl-Ab or a vehicle was administered accordingly in the groups. After seven days, all rats underwent the first lower molar extraction and were euthanized 15 days later. Computed microtomography, histological analysis, and collagen content measurement were performed on post-extraction sockets and intact mandibular and maxillary bone areas. ResultsMicrotomographic analyses of the sockets and mandibles did not reveal significant differences between groups on bone morphometric parameters (p > 0.05), while maxillary bone analyses resulted in better maintenance of bone architecture in OST+Scl-Ab, compared to OST (p < 0.05). Descriptive histological analysis and polarization microscopy indicated better post-extraction socket repair characteristics and collagen content in OST+Scl-Ab compared to OST (p < 0.05). ConclusionsScl-Ab-based medication did not accelerate alveolar bone formation but exhibited better post-extraction repair characteristics, and collagen content compared to ovariectomized animals only.

Development of a new method for the determination of 4-hydroxyproline as a measurement of collagen content in meat products and dietary supplements by cyclodextrin-assisted electrokinetic chromatography

The rise in consumption of ultra-processed meat and dietary collagen supplements challenges food quality control. 4-hydroxyproline stands out as collagen signature amino acid, and its determination is a well-known way to estimate collagen content, although for this measurement, the majority of the methods require a mandatory derivatization step. Therefore, this study aimed to develop an innovative method using cyclodextrin-assisted electrokinetic chromatography with indirect UV detection to determine 4-hydroxyproline for collagen monitoring in dietary supplements and meat products. Separations were performed in fused silica capillaries, at 12.5 °C, detection at 220 nm, 30 kV voltage, and hydrodynamic injection. Background electrolyte consisted of histidine 25 mmol L-1, butylamine 180 mmol L-1, and β-cyclodextrin 10 mmol L-1 at pH 11.4. Samples were hydrolyzed with sulfuric acid, and their removal was accomplished by simultaneous neutralization/precipitation reactions using strontium carbonate. Validation assured method detectability, accuracy (recoveries 88% - 102%), and precision (CV < 7%). Dietary supplements and meat products were successfully evaluated, indicating that this method is a feasible quality control alternative.

Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Inhibitor as a Novel Therapeutic Tool for Lung Injury.

Pulmonary fibrosis is a progressive disease characterized by lung remodeling due to excessive deposition of extracellular matrix. In this study, the bleomycin experimental model of pulmonary fibrosis was employed to investigate the anti-fibrotic and immunomodulatory activity of the inhibition of MALT1 protease activity. Mice received a single intra-tracheal administration of bleomycin (1 mg/kg) in the presence or absence of MI-2, a selective MALT1 inhibitor, (a dose of 30 mg/kg administered intra-peritoneally 1 h after bleomycin and daily until the end of the experiment). Seven days after bleomycin instillation mice were sacrificed and bronchoalveolar lavage fluid analysis, measurement of collagen content in the lung, histology, molecular analysis and immunohistochemistry were performed. To evaluate mortality and body weight gain a subset of mice was administered daily with MI-2 for 21 days. Mice that received MI-2 showed decreased weight loss and mortality, inflammatory cells infiltration, cytokines overexpression and tissue injury. Moreover, biochemical and immunohistochemical analysis displayed that MI-2 was able to modulate the excessive production of reactive oxygen species and the inflammatory mediator upregulation induced by bleomycin instillation. Additionally, MI-2 demonstrated anti-fibrotic activity by reducing transforming growth factor-β (TGF-β), α-smooth muscle actin (α-SMA) and receptor associated factor 6 (TRAF6) expression. The underlying mechanisms for the protective effect of MI-2 bleomycin induced pulmonary fibrosis may be attributed to its inhibition on NF-κB pathway. This is the first report showing the therapeutic role of MALT1 inhibition in a bleomycin model of pulmonary fibrosis, thus supporting further preclinical and clinical studies.

Intramuscular Anatomy Drives Collagen Content Variation Within and Between Muscles.

The passive load bearing properties of muscle are poorly understood partly due to challenges in identifying the connective tissue structures that bear loads. Prior attempts to correlate passive mechanical properties with collagen content (often expressed as a mass ratio and used as a surrogate for connective tissue quantity within muscle) have not been successful. This is likely a result of not accounting for variability in intramuscular connective tissue throughout a muscle such that a single collagen content value likely does not adequately represent the connective tissue load bearing capacity of a muscle. Therefore, the purpose of this study was to determine how intramuscular connective tissue distribution throughout a muscle impacts measured collagen content. For this analysis, four mouse hindlimb muscles were chosen because of their varying actions and anatomy; rectus femoris, semimembranosus, tibialis anterior, and lateral gastrocnemius. Collagen content throughout each muscle was determined biochemically using an optimized hydroxyproline assay. Dense connective tissue distribution throughout each muscle’s length was quantified histologically. We found that collagen content varied widely within and between muscles, from 3.6 ± 0.40 SEM μg/mg wet weight to 15.6 ± 1.58 SEM μg/mg, which is dependent on both the specific location within a muscle and particular muscle studied. Both collagen content and connective tissue structures demonstrated stereotypically patterns with the highest quantity at the proximal and distal ends of the muscles. Additionally, using three independent approaches: (1) linear regression, (2) predictive modeling, and (3) non-linear optimization, we found complementary and corroborating evidence suggesting a causal relationship between a muscle’s connective tissue distribution and collagen content. Specifically, we found that muscle collagen content is driven primarily by its dense connective tissue structures due to the extremely high collagen content of connective tissue (227.52–334.69 μg/mg) compared to muscle tissue (1.93–4.03 μg/mg). A consequence of these findings is that a single collagen content measurement does not accurately represent a muscle’s complex distribution of connective tissue. Future studies should account for collagen content variations and connective tissue anatomy to establish more accurate relationships between collagen content measurements and whole muscle passive mechanics.

Detection of collagens by multispectral optoacoustic tomography as an imaging biomarker for Duchenne muscular dystrophy.

Biomarkers for monitoring of disease progression and response to therapy are lacking for muscle diseases such as Duchenne muscular dystrophy. Noninvasive in vivo molecular imaging with multispectral optoacoustic tomography (MSOT) uses pulsed laser light to induce acoustic pressure waves, enabling the visualization of endogenous chromophores. Here we describe an application of MSOT, in which illumination in the near- and extended near-infrared ranges from 680-1,100 nm enables the visualization and quantification of collagen content. We first demonstrated the feasibility of this approach to noninvasive quantification of tissue fibrosis in longitudinal studies in a large-animal Duchenne muscular dystrophy model in pigs, and then applied this approach to pediatric patients. MSOT-derived collagen content measurements in skeletal muscle were highly correlated to the functional status of the patients and provided additional information on molecular features as compared to magnetic resonance imaging. This study highlights the potential of MSOT imaging as a noninvasive, age-independent biomarker for the implementation and monitoring of newly developed therapies in muscular diseases.

Transmembrane protease, serine 4 (TMPRSS4) is upregulated in IPF lungs and increases the fibrotic response in bleomycin-induced lung injury.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease characterized by epithelial cell activation, expansion of the fibroblast population and excessive extracellular matrix accumulation. The mechanisms are incompletely understood but evidence indicates that the deregulation of several proteases contributes to its pathogenesis. Transmembrane protease serine 4 (TMPRSS4) is a novel type II transmembrane serine protease that may promote migration and facilitate epithelial to mesenchymal transition (EMT), two critical processes in the pathogenesis of IPF. Thus, we hypothesized that over-expression of TMPRSS4 in the lung could promote the initiation and/or progression of IPF. In this study we first evaluated the expression and localization of TMPRSS4 in IPF lungs by real time PCR, western blot and immunohistochemistry. Then we examined the lung fibrotic response in wild-type and TMPRSS4 deficient mice using the bleomycin-induced lung injury model. We found that this protease is upregulated in IPF lungs, where was primarily expressed by epithelial and mast cells. Paralleling the findings in vivo, TMPRSS4 was expressed by alveolar and bronchial epithelial cells in vitro and unexpectedly, provoked an increase of E-cadherin. No expression was observed in normal human or IPF lung fibroblasts. The lung fibrotic response evaluated at 28 days after bleomycin injury was markedly attenuated in the haplodeficient and deficient TMPRSS4 mice. By morphology, a significant reduction of the fibrotic index was observed in KO and heterozygous mice which was confirmed by measurement of collagen content (hydroxyproline: WT: 164±21.1 μg/lung versus TMPRSS4 haploinsufficient: 110.2±14.3 μg/lung and TMPRSS4 deficient mice: 114.1±24.2 μg/lung (p<0.01). As in IPF, TMPRSS4 was also expressed in epithelial and mast cells. These findings indicate that TMPRSS4 is upregulated in IPF lungs and that may have a profibrotic role.

Zonal variation of MRI-measurable parameters classifies cartilage degradation

Osteoarthritis (OA) is a degenerative joint disease resulting in the deterioration of articular cartilage, a tissue with minimal ability to self-repair. Early diagnosis of OA with non-invasive imaging techniques such as magnetic resonance imaging (MRI) could provide an opportunity to intervene and slow or reverse this degeneration process. This study examines the classification of degradation states using MRI measurements.Enzymatic degradation was used to specifically target proteoglycans alone, collagen alone and both cartilage components sequentially. The resulting degradation was evaluated using MRI imaging techniques (T1, T2, diffusion tensor imaging, and gadolinium enhanced T1) and derived measures of water, glycosaminoglycan and collagen content. We compared the classification ability of full thickness averages of these parameters with zonal averages (superficial, medial, and deep). Finally, we determined minimum variables sets to identify the smallest number of variables that allowed for complete separation of all degradation groups and ranked them by impact on the separation.Zonal analysis was much more sensitive than full thickness averages and allowed perfect separation of all four groups. Superficial zone cartilage was more sensitive to enzymatic degradation than the medial or deep zone, or the full thickness average. Variable ranking consistently identified collagen content and organization as the most impactful variables in the classification algorithm.The aim of this study is to classify cartilage degradation using only non-invasive MRI parameters that could be applied to OA diagnosis. Our results highlight the importance of zonal variation in the diagnosis of cartilage degeneration. Our novel, non-invasive collagen content measurement was crucial for complete separation of degraded groups from control cartilage. These findings have significant implications for clinical cartilage MRI for disease diagnosis.

Epoxyeicosatrienoic Acid Analog Decreases Renal Fibrosis by Reducing Epithelial-to-Mesenchymal Transition.

Renal fibrosis, which is a critical pathophysiological event in chronic kidney diseases, is associated with renal epithelial-to-mesenchymal transition (EMT). Epoxyeicosatrienoic acids (EETs) are Cyp epoxygenase arachidonic acid metabolites that demonstrate biological actions that result in kidney protection. Herein, we investigated the ability of 14,15-EET and its synthetic analog, EET-A, to reduce kidney fibrosis induced by unilateral ureter obstruction (UUO). C57/BL6 male mice underwent sham or UUO surgical procedures and were treated with 14,15-EET or EET-A in osmotic pump (i.p.) for 10 days following UUO surgery. UUO mice demonstrated renal fibrosis with an 80% higher kidney-collagen positive area and 70% higher α-smooth muscle actin (SMA) positive renal areas compared to the sham group. As a measure of collagen content, kidney hydroxyproline content was also higher in UUO (6.4 ± 0.5 μg/10 mg) compared to sham group (2.5 ± 0.1 μg/10 mg). Along with marked renal fibrosis, UUO mice had reduced renal expression of EET producing Cyp epoxygenase enzymes. Endogenous 14,15-EET or EET-A demonstrated anti-fibrotic action in UUO by reducing kidney-collagen positive area (50–60%), hydroxyproline content (50%), and renal α-SMA positive area (85%). In UUO mice, renal expression of EMT inducers, Snail1 and ZEB1 were higher compared to sham group. Accordingly, renal epithelial marker E-cadherin expression was reduced and mesenchymal marker expression was elevated in the UUO compared to sham mice. Interestingly, EET-A reduced EMT in UUO mice by deceasing renal Snail1 and ZEB1 expression. EET-A treatment also opposed the decrease in renal E-cadherin expression and markedly reduced several prominent renal mesenchymal/myofibroblast markers in UUO mice. Overall, our results demonstrate that EET-A is a novel anti-fibrotic agent that reduces renal fibrosis by decreasing renal EMT.

The effect of angoroside C on pressure overload-induced ventricular remodeling in rats

BackgroundOur previous study reveals that total rough extract of Radix Scrophulariae has a beneficial effect on ventricular remodeling. HypothesisAfter carrying out a series of preliminary experiments, we speculated that angoroside C may be the effective agent. Study designAfter oral administration, the effect of angoroside C on ventricular remodeling was evaluated by using a pressure-overloaded rat model, some related indexes were detected in vivo. MethodsA model of pressure overloaded ventricular remodeling was produced by abdominal aortic constriction (AAC) in rats. The sham-operated rats underwent an identical surgical procedure except for AAC. AAC rats were randomly divided into five groups: model control group, three angoroside C treated groups (7.5, 15 and 30 mg·kg−1) and captopril treated group (40 mg·kg−1). The rats were orally administered with the corresponding drugs or drinking water for 4 weeks. The levels of blood pressure (BP), left ventricular weight index (LVWI) and heart weight index (HWI) were detected. Myocardium tissue was stained with hematoxylin and eosin or picric acid/sirius red for cardiomyocyte cross-section area or collagen content measurements respectively. The concentrations of angiotensin Ⅱ (Ang Ⅱ), hydroxyproline (Hyp), matrix metalloproteinase 2 (MMP-2), MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in myocardium or serum were determined. Real-time RT-PCR was performed to detect the mRNA expressions of endothelin 1 (ET-1), transforming growth factor β1 (TGF-β1). ResultsAngoroside C significantly reduced the BP, LVWI and HWI, decreased the content of Ang Ⅱ, Hyp, diminished cross sectional area of cardiomyocytes and ameliorated collagen deposition. Additionally, it markedly reduced collagen I and III expressions and regulated matrix metalloproteinase-2, 9 and inhibitors of metalloproteinase expressions. Angoroside C also down regulated the gene expressions of ET-1 and TGF-β1mRNA in myocardium. ConclusionAngoroside C has beneficial effects against ventricular remodeling. The mechanism is likely to be related to decreasing the level of Ang Ⅱ, attenuating the mRNA expressions of ET-1 and TGF-β1.

Beneficial effects of houttuynin on ventricular remodeling induced by coronary artery ligation in rats

To examine the effects of houttuynin on ventricular remodeling induced by coronary artery ligation in rats and the underlying mechanisms. A rat model of ventricular remodeling was established by left coronary artery ligation (CAL). Rats were randomly divided into four groups: CAL control, CAL plus 40mg/kg captopril, CAL plus 100mg/kg houttuynin and sham-operated control. The rats were administered intragastrically with the corresponding drugs or distilled water for 7 weeks. At the end of the experiment, the left ventricular weight index (LVWI) and heart weight index (HWI) were determined. Myocardium tissue was stained with hematoxylin and eosin or picric acid/Sirius red for cardiomyocyte cross-section area or collagen content measurements respectively. The concentrations of angiotensin I (Ang I), angiotensin II (Ang II), aldosterone (ALD) and endothelin-1 (ET-1) in myocardium or serum were detected by radioimmunoassay. The hydroxyproline (Hyp) concentration was measured by alkali hydrolysis. Ultraviolet spectrophotometry was used to determine glutathione peroxidase (GSH-Px) and catalase (CAT) activities in serum. Houttuynin significantly diminished LVWI and HWI, decreased Ang I, Ang II, ALD, ET-1 and Hyp concentrations in myocardium or serum, increased NO concentration and GSH-Px, CAT activities after 7 weeks of treatment. Houttuynin could also reduce cardiomyocyte cross-section area and collagen deposition. Houttuynin attenuates ventricular remodeling in coronary artery ligation rats by restricting the excessive activation of rennin-angiotensin-aldosterone system (RAAS) and the peroxidation.

Thymosin β4 reduces IL-17-producing cells and IL-17 expression, and protects lungs from damage in bleomycin-treated mice

Thymosin β4 (Tβ4) is a highly conserved peptide with immunomodulatory properties. In this research we investigated the effects of Tβ4 on the bleomycin-induced lung damage in CD-1 mice and the changes in the number of IL-17-producing cells as well as the IL-17 expression in the lung. Male CD-1 mice were treated with bleomycin (1mg/kg) in the absence or the presence of Tβ4 (6mg/kg delivered intra-peritoneally on the day of bleomycin treatment and for 2 additional doses). After sacrifice one week later, lung histology, measurement of collagen content of the lung, Broncho Alveolar Lavage Fluid (BALF) analysis, evaluation of IL17-producing cells in the blood as well as RT-PCR and IHC in the lung tissue were performed.As expected, bleomycin-induced inflammation and lung damage were substantially reduced by Tβ4 treatment in CD-1 mice, as shown by the significant reduction of (i) leukocytes in BALF, (ii) histological evidence of the lung damage, and (iii) total collagen content in the lung. Importantly, the bleomycin-induced increase in the number of IL17-producing cells in the blood was significantly blocked by Tβ4. Accordingly, IHC and RT-PCR results demonstrated that Tβ4 substantially inhibited bleomycin-induced IL-17 over-expression in the lung tissue.This is the first report showing that a decreased amount of IL17-producing cells and inhibited IL-17 expression in the lung with Tβ4 treatment correlate with its anti-inflammatory and anti-fibrotic effects.

The Use of Collagen Content as Determined by Spectral Domain Polarization-Sensitive Optical Coherence Tomography to Assess Colon Anastomosis Healing in a Rat Model

Background/Purpose: Many studies have been undertaken to prevent anastomosis leakage of the colon, and several methods have been used to assess anastomosis healing, such as measurement of bursting pressure or hydroxyproline (a marker of collagen) content at the anastomosis site. However, these methods are inappropriate for comparing anastomosis healing at two time points in the same animals. In the present study, we measured the collagen level by spectral domain polarization-sensitive optical coherence tomography (SD-PS-OCT) to assess anastomosis healing. Methods: Sprague-Dawley rats were divided into groups C (saline-administered controls; study group) and M [a 5-fluorouracil (5-FU)-administered experimental group]. Immediately after end-to-end anastomosis of the colon, SD-PS-OCT images of anastomoses were taken (baseline). Animals were administered saline or 5-FU for 7 days. On the 7th postoperative day, SD-PS-OCT images were acquired, a histopathologic exam was performed, and hydroxyproline levels as well as mRNA expressions of collagen-1 and collagen-3 were measured at the anastomosis site. Results: Fibroblast proliferation and inflammatory cell infiltration were greater in group C than in group M. The mRNA expressions of collagen-1 and collagen-3 were substantially higher in group C. Hydroxyproline levels were higher in group M than in group C. Though collagen levels measured by SD-PS-OCT at 7 days were elevated compared with baseline in group C, no such changes were observed for group M. Conclusion: Collagen levels at the colon anastomosis site, measured with SD-PS-OCT, were not increased at 7 days postoperatively versus baseline when 5-FU was injected, but were increased in saline-treated controls. The measurement of collagen content by SD-PS-OCT was found to provide a good means of assessing anastomosis healing, because it allows in situ assessment of collagen contents at baseline and during the postoperative period.

Conditioned medium from amniotic membrane-derived cells prevents lung fibrosis and preserves blood gas exchanges in bleomycin-injured mice—specificity of the effects and insights into possible mechanisms

Background and aimsWe recently demonstrated that injection of conditioned medium (CM) generated from cells of the mesenchymal region of human amniotic membrane (AMTCs) reduces bleomycin-induced lung fibrosis in mice, suggesting a crucial role of paracrine factor(s) secreted by AMTCs in these beneficial effects. We further investigated this hypothesis, the mechanisms involved, the effects on some lung functional parameters and whether AMTC-secreted effector(s) are specific to these cells and not produced by other cell types, extending the time of analysis up to 28 days after treatment. MethodsBleomycin-challenged mice were either treated with AMTC-CM or CM generated from human skin fibroblasts, human peripheral blood mononuclear cells or Jurkat cells, or were left untreated. Mouse lungs were analyzed for content of pro-inflammatory and pro-fibrotic molecules, presence of lymphocytes and macrophages and for fibrosis level (through histological semi-quantitative evaluation and quantitative measurement of collagen content). Arterial blood gas analysis was also performed. ResultsUp to 28 days after delivery, AMTC-CM-treated mice developed reduced lung fibrosis with respect to mice treated with other CM types. AMTC-CM-treated mice had comparatively better preservation of blood gas parameters and showed lower lung content of interleukin-6, tumor necrosis factor-α, macrophage inflammatory protein-1α, monocyte chemoattractant protein-1 and transforming growth factor-β associated with reduced lung macrophage levels. ConclusionsAMTC-CM prevents lung fibrosis in bleomycin-challenged mice, improving survival and preserving lung functional parameters such as blood gas exchanges. The specificity of AMTC-CM action was indicated by the absence of fibrosis reduction when other CM types were used. Finally, we provide some insights into the possible mechanisms underlying AMTC-CM-mediated control of fibrosis.

Abstract 317: Endoplasmic Reticulum Stress Contributes to Aortic Stiffening Through Increasing Collagen Synthesis and Deposition

Aortic stiffening is an independent predictor of negative cardiovascular outcomes and is associated with an increased risk of hypertension. Aortic fibrosis contributes to stiffening through the synthesis and deposition of collagen within the vessel wall. Endoplasmic reticulum (ER) stress has been linked with cardiac fibrosis and in non-vascular cell types ER stress can trigger collagen synthesis. However, the role of ER stress in aortic fibrosis and collagen synthesis remains unclear. We hypothesized that ER stress promotes aortic fibrosis through increasing collagen synthesis and deposition. In vitro, collagen synthesis was measured in the media from primary aortic vascular smooth muscle cells (VSMCs, third passage) treated at different time points (0, 6, 12, 24 hrs) with ER stress inducer, tunicamycin (TM, 1 μg/ml) using Sirius red and analyzed with a spectrophotometer (ODN 550 nM). In vivo, male Sprague-Dawley rats (12 wks) were implanted with mini-osmotic pumps containing TM (10 μg/kg/day, 14 days) or received sham surgery. Thoracic aorta was used for Western blot (data presented as fold change from control ± SEM) and immunohistochemical staining with Sirius red, a measure of collagen content and Masson’s trichrome stain, a measurement of fibrosis. Systolic blood pressure was increased in rats treated with TM compared to control (mmHg; 167±2 vs. 128±3). There was an increase in aortic wall thickness of the TM treated rats compared to control (mm; 0.35±0.01 vs. 0.32±0.01). Aortic expression of known proteins upregulated following induction of ER stress were increased by TM treatment: GRP78, an ER chaperone (1.4±0.3), PDI, a protein disulfide isomerase (1.3±0.2) and CHOP, a pro-apoptotic protein (1.7±0.3). Treatment with TM increased both aortic collagen content (% area; 10.7±0.5 vs. 7.4±0.4) and fibrosis (% area; 10.4±0.4 vs. 5.1±0.9) compared to control. There was a time-dependent increase in collagen synthesis from the media of VSMCs cells treated with TM (ODN 550nM: 0hrs, 0.9±0.002; 6hrs, 0.12±0.004; 12hrs, 0.17±0.005; 24hrs, 0.28±0.008). Our data suggest that ER stress may contribute to aortic stiffening through increasing collagen synthesis and deposition.

Cellular Mechanisms of Tissue Fibrosis. 4. Structural and functional consequences of skeletal muscle fibrosis

Skeletal muscle fibrosis can be a devastating clinical problem that arises from many causes, including primary skeletal muscle tissue diseases, as seen in the muscular dystrophies, or it can be secondary to events that include trauma to muscle or brain injury. The cellular source of activated fibroblasts (myofibroblasts) may include resident fibroblasts, adult muscle stem cells, or inflammatory or perivascular cells, depending on the model studied. Even though it is likely that there is no single source for all myofibroblasts, a common mechanism for the production of fibrosis is via the transforming growth factor-β/phosphorylated Smad3 pathway. This pathway and its downstream targets thus provide loci for antifibrotic therapies, as do methods for blocking the transdifferentiation of progenitors into activated fibroblasts. A structural model for the extracellular collagen network of skeletal muscle is needed so that measurements of collagen content, morphology, and gene expression can be related to mechanical properties. Approaches used to study fibrosis in tissues, such as lung, kidney, and liver, need to be applied to studies of skeletal muscle to identify ways to prevent or even cure the devastating maladies of skeletal muscle.

Effect of oxymatrine, the active component from Radix Sophorae flavescentis (Kushen), on ventricular remodeling in spontaneously hypertensive rats

PurposeTo examine the effects of oxymatrine (OMT) on ventricular remodeling in spontaneous hypertension rat (SHR) and the underlying mechanism. MethodsSHRs were divided into four groups: SHR control, SHR+40mg/kg captopril, SHR+30mg/kg OMT and SHR+60mg/kg OMT. Normotensive age-matched WKY rats were assigned to two groups: WKY control, WKY+30mg/kg OMT. The rats were orally administered with the corresponding drugs or drinking water for 21 weeks. Mean arterial blood pressure (MAP) and heart rate (HR) were measured. The left ventricular weight index (LVWI) and heart weight index (HWI) were determined. Myocardium tissue was stained with picric acid/Sirius red for measurement of collagen content measurements. The concentrations of serum norepinephrine and angiotensin II (Ang II) in myocardium were determined. Real-time RT-PCR was used to detect the mRNA expressions of transforming growth factor-β1 (TGF-β1), collagen types I, III and angiotensin converting enzyme (ACE). Western blots were performed to determine bioactivities of extracellular signal regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK/SAPK), p38 mitogen-activated protein kinases (p38 MAPK) and phospho-specific protein kinase C (PKC). ResultsIn the SHR, hypertension, myocardium hypertrophy, more cardiac fibrosis, higher concentrations of serum norepinephrine and myocardium Ang II were observed. OMT treatment lowered the blood pressure, reduced the concentrations of serum norepinephrine and myocardium Ang II, favorably decreased the measured gravimetric parameters, decreased the interstitial and perivascular collagen deposition, attenuated the collagen of type I and III accumulation, downregulated the mRNA expression of ACE and TGF-β1, and suppressed the phosphorylation of ERK 1/2, JNK and p38 MAPK in SHRs. ConclusionOMT prevents ventricular remodeling in SHR. The mechanisms may be related to inhibiting the gene overexpression of ACE and TGF-β1, suppressing the activation of signaling pathways of ERK 1/2, JNK and p38 MAPK.

An experimental model for the study of collagen fibers in skeletal muscle

To examine histological and histomorphometric techniques for measuring collagen in skeletal muscle. The following staining methods were used in the study: hematoxylin and eosin, Masson's trichrome, reticulin, and picrosirius red, and immunostaining for collagen types I, II, III, IV, and V. Histomorphometric measurements were performed using Corel PhotoPaint and UTHSCSA Image Tool 3.0 software. Both the Masson's trichrome and picrosirius red staining provided the best visualization for the measurement of collagen content. This methodology is important for the identification and quantification of the different types of collagen in muscles and can be used in the investigation of the qualitative and quantitative influence of collagen on physical activities, aging, and diseases.

Development and evaluation of a decellularized membrane from human dermis

Interest is increasing in biological scaffolds for tissue regeneration, such as extracellular matrix (ECM) membranes, developed through soft tissue decellularization. The present study describes the development of a chemicophysical decellularization method applied to allogenic human-derived dermis (HDM). To evaluate the absence of viable cells and the maintenance of ECM structure, biological, histological and ultrastructural assessments were performed on the HDM membrane. Residual DNA content and glycosaminoglycan (GAG) and collagen contents were quantified. Growth factor (GF) release was directly measured on HDM extracts and indirectly measured by assessing cell proliferation after administering extract to cultures. Tensile tests were performed to measure the effect of the decellularization technique on the mechanical properties of tissue. Histocompatibility was investigated after subcutaneous implantation in rats. Residual DNA, GAG and collagen content measurements, vitality index, histology and electron microscopy showed the efficiency of the decellularization process and preservation of ECM matrix and bioactivity. In HDM extracts, among the tested GFs, transforming growth factor-β1 showed the highest concentration. HDM extracts significantly increased the proliferation rate of L929 fibroblasts in comparison with controls (p < 0.005, p < 0.05 and p < 0.0005). Maximum load and stiffness of HDM were significantly higher than those of cellularized dermis (p < 0.0005, p < 0.005). Histological and histomorphometric analysis of explanted samples showed that the membrane was integrated with host tissues in the absence of inflammatory reactions. Our results show that the decellularization method allowed the development of a human allograft dermal matrix that might be useful for soft tissue regeneration.

Quantitative analysis of estimated scattering coefficient and phase retardation for ovarian tissue characterization

In this report, optical scattering coefficient and phase retardation quantitatively estimated from polarization-sensitive OCT (PSOCT) were used for ovarian tissue characterization. A total of 33 ex vivo ovaries (normal: n = 26, malignant: n = 7) obtained from 18 patients were investigated. A specificity of 100% and a sensitivity of 86% were achieved by using estimated scattering coefficient alone; and a specificity of 100% and a sensitivity of 43% were obtained by using phase retardation alone. However, a superior specificity of 100% and sensitivity of 100% were achieved if these two parameters were used together for classifying normal and malignant ovaries. Quantitative measurement of collagen content obtained from Sirius red histology sections shows that it correlates with estimated scattering coefficient and phase retardation. Our initial results demonstrate that quantitative analysis of PSOCT could be a potentially valuable method for distinguishing normal from malignant ovarian tissues during minimally invasive surgery and help guide surgical intervention.

Angiotensin II induces skin fibrosis: a novel mouse model of dermal fibrosis

IntroductionSystemic sclerosis (SSc) is an autoimmune inflammatory disorder of unknown etiology characterized by fibrosis of the skin and internal organs. Ang II (angiotensin II), a vasoconstrictive peptide, is a well-known inducer of kidney, heart, and liver fibrosis. The goal of this study was to investigate the profibrotic potential of Ang II in the mouse skin.MethodsAng II was administered by subcutaneous osmotic mini pumps to C57BL/6 male mice. Collagen-content measurements were performed with Gomori Trichrome staining and hydroxyproline assay. The mRNA expression level of collagens, TGF-β1, TGF-β2, TGF-β3, CTGF, αSMA, CD3, Emr1, CD45/B220, MCP1, and FSP1 were quantified with real-time polymerase chain reaction (PCR). Immunostaining was performed for markers of inflammation and fibrosis, including, phospho-Smad2, αSMA, CD3, Mac3, CD45/B220, and CD163B. Fibrocytes were identified by double staining with CD45/FSP1 and CD45/PH4. Endothelial cells undergoing endothelial-to-mesenchymal transition (EndoMT) were identified by double staining with VE-cadherin/FSP1.ResultsAng II-infused mice develop prominent dermal fibrosis in the area proximal to the pump, as shown by increased collagen and CTGF mRNA levels, increased hydroxyproline content, and more tightly packed collagen fibers. In addition, elevated mRNA levels of TGF-β2 and TGF-β3 along with increased expression of pSmad2 were observed in the skin of Ang II-treated mice. Dermal fibrosis was accompanied by an increased number of infiltrating fibrocytes, and an increased number of αSMA-positive cells, as well as CD163B+ macrophages in the upper dermis. This correlated with significantly increased mRNA levels of αSMA, Emr1, and MCP1. Infiltration of CD3-, CD45/B220-, and Mac3-positive cells was observed mainly in the hypodermis. Furthermore, an increased number of double-positive VE-cadherin/FSP1 cells were detected in the hypodermis only.ConclusionsThis work demonstrates that Ang II induces both inflammation and fibrosis in the skin via MCP1 upregulation and accumulation of activated fibroblasts. Additionally, our data suggest that populations of these fibroblasts originate from circulating blood cells. Ang II infusion via osmotic minipumps could serve as a useful mouse model of skin fibrosis to gain new insights into pathogenic mechanisms and to test new antifibrotic therapies.