Measure Collagen Content Research Articles

Arsenic trioxide demonstrates efficacy in a mouse model of preclinical systemic sclerosis

BackgroundUncontrolled T-cell activation plays a key role in systemic sclerosis (SSc). Arsenic trioxide (ATO) has immunological effects and has demonstrated potential in preclinical SSc models. In this study, we assessed the efficacy of ATO in Fra2 transgenic (Fra2TG) mice, which develop severe vascular remodeling of pulmonary arterioles and nonspecific interstitial pneumonia-like lung disease, closely resembling human SSc-associated pulmonary hypertension, therefore partially resembling to the SSc human disease.MethodsThe efficacy of ATO in Fra2TG mice was evaluated through histological scoring and determination of cell infiltration. Fibrotic changes in the lungs were assessed by measuring collagen content biochemically, using second harmonic generation to measure fibrillar collagen, and imaging via computed tomography. Cardiovascular effects were determined by measuring right ventricular systolic pressure and vessel remodeling. The mechanism of action of ATO was then investigated by analyzing lung cell infiltrates using flow cytometry and bulk RNA with sequencing techniques.ResultsAfter ATO treatment, the Ashcroft histological score was substantially decreased by 33% in ATO-treated mice compared to control mice. Other investigations of fibrotic markers showed a trend of reduction in various measurements of fibrosis, but the differences did not reach significance. Further cardiovascular investigations revealed convergent findings supporting a beneficial effect of ATO, with reduced right ventricular systolic pressure and medial wall thickness, and a significant decrease in the number of muscularized distal pulmonary arteries in ATO-treated Fra2TG mice compared to untreated Fra2TG mice. Additionally, inflammatory cell infiltration was also markedly reduced in lesioned lungs. A reduction in the frequency of CD4 + and T effector memory cells, and an increase in the percentage of CD4 + T naive cells in the lungs of ATO-treated Fra-2TG mice, was observed when compared to PBS group Fra-2Tg mice. RNA-seq analysis of ATO-treated mouse lungs revealed a downregulation of biological pathways associated with immune activity and inflammation, such as T-cell activation, regulation of leucocyte activation, leucocyte cell–cell adhesion, and regulation of lymphocyte activation.ConclusionsOur results suggest the clinical relevance of ATO treatment in SSc. Using the Fra2TG mouse model, we observed significant lung histological changes, a trend towards a decrease in various fibrotic makers, and a strong reduction in vascular remodeling. The mechanism of action of ATO appears to involve a marked counteraction of the immune activation characteristic of SSc, particularly T-cell involvement. These findings pave the way for further studies in SSc.

Doxorubicin Dose-Dependent Impact on Physiological Balance-A Holistic Approach in a Rat Model.

Doxorubicin (DOX) is commonly used in several chemotherapies to treat various cancers, but it is known to cause cardiotoxicity and cardiac symptoms. Autonomic dysfunction is thought to contribute to the cardiotoxic effects of DOX, but the specific dose required to disrupt homeostatic processes is still unclear and is influenced by numerous factors. This study aimed to investigate how the DOX dosage affects autonomic function and physiological parameters, to elucidate the neurocardiac mechanisms underlying the observed cardiovascular side effects. Wistar rats were treated with DOX for four weeks and divided into three dosing groups: DOX8 (2 mg/kg/week), DOX16 (4 mg/kg/week), and DOX20 (5 mg/kg/week). A control group received NaCl 0.9% saline (1 mL/kg/week). In an acute experiment, we recorded blood pressure (BP), electrocardiogram, heart rate (HR), and respiratory rate (RF). Baroreflex gain and chemoreflex sensitivity were calculated, and cardiac tissue was analyzed with picrosirius histochemistry to measure collagen content. Our results showed that the LF/HF ratio, indicative of autonomic activity, was altered along with hypotension and bradycardia at a cumulative DOX dose threshold of 16 mg/kg. We observed a positive correlation between DOX dose and BP, HR, urinary norepinephrine, LF/HF ratio, and fibrotic heart area. Lower LF/HF ratios were associated with high DOX doses, reflecting drug-induced impairment of autonomic control of HR. This study provides valuable insights into the dose-dependent effects of DOX on physiological parameters and the development of cardiovascular dysfunction. These findings are critical, which is important for optimizing the management and therapeutic strategies for patients undergoing DOX-based chemotherapy.

Phosphotungstic acid (PTA) preferentially binds to collagen- rich regions of porcine carotid arteries and human atherosclerotic plaques observed using contrast enhanced micro-computed tomography (CE-µCT).

Background and aims: Atherosclerotic plaque rupture in the carotid artery can cause small emboli to travel to cerebral arteries, causing blockages and preventing blood flow leading to stroke. Contrast enhanced micro computed tomography (CEμCT) using a novel stain, phosphotungstic acid (PTA) can provide insights into the microstructure of the vessel wall and atherosclerotic plaque, and hence their likelihood to rupture. Furthermore, it has been suggested that collagen content and orientation can be related to mechanical integrity. This study aims to build on existing literature and establish a robust and reproducible staining and imaging technique to non-destructively quantify the collagen content within arteries and plaques as an alternative to routine histology. Methods: Porcine carotid arteries and human atherosclerotic plaques were stained with a concentration of 1% PTA staining solution and imaged using MicroCT to establish the in situ architecture of the tissue and measure collagen content. A histological assessment of the collagen content was also performed from picrosirius red (PSR) staining. Results: PTA stained arterial samples highlight the reproducibility of the PTA staining and MicroCT imaging technique used with a quantitative analysis showing a positive correlation between the collagen content measured from CEμCT and histology. Furthermore, collagen-rich areas can be clearly visualised in both the vessel wall and atherosclerotic plaque. 3D reconstruction was also performed showing that different layers of the vessel wall and various atherosclerotic plaque components can be differentiated using Hounsfield Unit (HU) values. Conclusion: The work presented here is unique as it offers a quantitative method of segmenting the vessel wall into its individual components and non-destructively quantifying the collagen content within these tissues, whilst also delivering a visual representation of the fibrous structure using a single contrast agent.

Tissue Rigidity Increased during Carcinogenesis of NTCU-Induced Lung Squamous Cell Carcinoma In Vivo.

Increased tissue rigidity is an emerging hallmark of cancer as it plays a critical role in promoting cancer growth. However, the field lacks a defined characterization of tissue rigidity in dual-stage carcinogenesis of lung squamous cell carcinoma (SCC) in vivo. Pre-malignant and malignant lung SCC was developed in BALB/c mice using N-nitroso-tris-chloroethylurea (NTCU). Picro sirius red staining and atomic force microscopy were performed to measure collagen content and collagen (diameter and rigidity), respectively. Then, the expression of tenascin C (TNC) protein was determined using immunohistochemistry staining. Briefly, all tissue rigidity parameters were found to be increased in the Cancer group as compared with the Vehicle group. Importantly, collagen content (33.63 ± 2.39%) and TNC expression (7.97 ± 2.04%) were found to be significantly higher (p < 0.05) in the Malignant Cancer group, as compared with the collagen content (18.08 ± 1.75%) and TNC expression (0.45 ± 0.53%) in the Pre-malignant Cancer group, indicating increased tissue rigidity during carcinogenesis of lung SCC. Overall, tissue rigidity of lung SCC was suggested to be increased during carcinogenesis as indicated by the overexpression of collagen and TNC protein, which may warrant further research as novel therapeutic targets to treat lung SCC effectively.

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.

Adaptations to Oral and Pharyngeal Swallowing Function Induced by Injury to the Mylohyoid Muscle.

Muscle injury is a frequent side effect of radiation treatment for head and neck cancer. To understand the pathophysiology of injury-related dysfunction, we investigated the effects of a single muscle injury to the mylohyoid on oropharyngeal swallowing function in the rat. The mylohyoid protects the airway from food/liquid via hyolaryngeal elevation and plays an active role during both oral and pharyngeal swallowing. We hypothesized (1) that fibrosis to the mylohyoid alters swallowing bolus flow and licking patterns and (2) that injury to the mylohyoid changes normal activity of submental, laryngeal, and pharyngeal muscles during swallowing. A chilled cryoprobe was applied to the rat mylohyoid muscle to create a localized injury. One and two weeks after injury, swallowing bolus transit was assessed via videofluoroscopy and licking behavior via an electrical lick sensor. The motor activity of five swallow-related muscles was analyzed immediately after injury using electromyography (EMG). Comparisons were made pre- and post-injury. Fibrosis was confirmed in the mylohyoid at 2 weeks after injury by measuring collagen content. One week after injury, bolus size decreased, swallowing rate reduced, and licking patterns were altered. Immediately post-injury, there was a significant depression in mylohyoid and thyropharyngeus EMG amplitudes during swallowing. Our results demonstrated that injury to the mylohyoid is sufficient to cause changes in deglutition. These disruptions in oral and pharyngeal swallowing were detected prior to long-term fibrotic changes, including delays in tongue movement, alterations in bolus flow, and changes in sensorimotor function. Therefore, injuring a single important swallowing muscle can have dramatic clinical effects.

Radial variation in biochemical composition of the bovine caudal intervertebral disc.

Bovine caudal discs have been widely used in spine research due to their increased availability, large size, and mechanical and biochemical properties that are comparable to healthy human discs. However, despite their extensive use, the radial variations in bovine disc composition have not yet been rigorously quantified with high spatial resolution. Previous studies were limited to qualitative analyses or provided limited spatial resolution in biochemical properties. Thus, the main objective of this study was to provide quantitative measurements of biochemical composition with higher spatial resolution than previous studies that employed traditional biochemical techniques. Specifically, traditional biochemical analyses were used to measure water, sulfated glycosaminoglycan, collagen, and DNA contents. Gravimetric water content was compared to data obtained through Raman spectroscopy and differential scanning calorimetry. Additionally, spatial distribution of lipids in the disc's collagen network was visualized and quantified, for the first time, using multi‐modal second harmonic generation (SHG) and Coherent anti‐Stokes Raman (CARS) microscopy. Some heterogeneity was observed in the nucleus pulposus, where the water content and water‐to‐protein ratio of the inner nucleus were greater than the outer nucleus. In contrast, the bovine annulus fibrosus exhibited a more heterogeneous distribution of biochemical properties. Comparable results between orthohydroxyproline assay and SHG imaging highlight the potential benefit of using SHG microscopy as a less destructive method for measuring collagen content, particularly when relative changes are of interest. CARS images showed that lipid deposits were distributed equally throughout the disc and appeared either as individual droplets or as clusters of small droplets. In conclusion, this study provided a more comprehensive assessment of spatial variations in biochemical composition of the bovine caudal disc.

556 Modeling tumor promoting extracellular matrix dynamics in 3-D suspension culture

Increased tissue stiffness, excessive extracellular matrix (ECM) densification and accumulation of collagen fibers and crosslinks are all associated with tumor cell invasion, immune evasion and tumor progression. We have developed a novel assay that recapitulates collagen fiber formation and tumor fibroblast collagen remodeling in vitro. Our method relies on tissue engineered from primary fibroblasts in suspension, and thus avoids the artificial mechanical stress associated with 2D culture on rigid substrates. Using this assay we measure collagen content and crosslinking via colorimetric analysis of total versus immature collagen fibers, relying upon collagen-specific Sirius red staining and analyses of birefringent collagen fibrillar deposits with the use of a polarizing microscope. Quantification of areas occupied by red-colored and green-colored fibrils is therefore a measure of collagen maturation and crosslinking, with red birefringence an indication of mature collagen fibrils and green birefringence an indication of immature collagen fibrils, as demonstrated by the effects of TGFb1 ligand stimulation and TGFb receptor type I inhibition. An increase of immature collagen fibrils over time is indicative of increased remodeling of collagen-rich ECM. Using multiple patient samples, we determined that collagen assemblies produced by squamous cell carcinoma (SCC) fibroblasts (n=4) are continually remodeled in contrast to those from isogenic normal fibroblasts (n=3), and that these differences correlate with invasion when fibroblast matrices are co-cultured with SCC keratinocytes. We are further investigating the parameters of collagen remodeling and tumor cell invasion with the use of b-aminopropionitrile, an irreversible inhibitor of lysyl oxidase, recombinant lysyl oxidase and the lysine analogue trans-4,5-dehydrolysine.

Quantitative Analysis of Collagen Produced by Rabbit Keratocytes using Second Harmonic Generation Microscopy

ABSTRACTPurpose: To quantitatively analyze the collagen produced by rabbit keratocytes treated with mitomycin C (MMC) and/or transforming growth factor-beta (TGF-β1), and to investigate the collagen arrangement in situ, using second harmonic generation (SHG) microscopy.Methods: Keratocytes were divided into four groups: The keraotocytes in group rabbit keratocytes were treated with serum free medium containing 1% methylcellulose as controls. The cells in group TGF were received with TGF-β1 (at 2 ng/ml) and the same medium as the control. After exposure to MMC at 0.02 mg/ml for 5 minutes, the cells in group MMC were received with the same treatment as controls. In group TGF-MMC, the cells were exposed to MMC at 0.02 mg/ml for 5 minutes first, and then treated as group TGF. The signals of collagen were detected by SHG at 2 and 4 weeks post-treatment. The collagen content, as well as the keratocyte numbers, was calculated by MetaMorph Offline.Results: The collagen arrangement could be detected clearly by SHG. The cell number of group TGF was higher than other groups at 2 and 4 weeks post-treatment. The collagen content in group MMC was the least among all the groups at 2 weeks, while the collagen content in group TGF was higher than in group rabbit keratocytes and MMC at 4 weeks. No significant SHG side effects were found in cell proliferation and collagen deposition.Conclusions: SHG can provide a highly sensitive method to quantitatively measure collagen content, as well as to analyze its arrangement in situ.

Sulforaphane effects on postinfarction cardiac remodeling in rats: modulation of redox-sensitive prosurvival and proapoptotic proteins

This study investigated whether sulforaphane (SFN), a compound found in cruciferous vegetables, could attenuate the progression of post-myocardial infarction (MI) cardiac remodeling. Male Wistar rats (350 g) were allocated to four groups: SHAM (n=8), SHAM+SFN (n=7), MI (n=8) and MI+SFN (n=5). On the third day after surgery, cardiac function was assessed and SFN treatment (5 mg/kg/day) was started. At the end of 25 days of treatment, cardiac function was assessed and heart was collected to measure collagen content, oxidative stress and protein kinase. MI and MI+SFN groups presented cardiac dysfunction, without signs of congestion. Sulforaphane reduced fibrosis (2.1-fold) in infarcted rats, which was associated with a slight attenuation in the cardiac remodeling process. Both infarcted groups presented increases in the oxidative markers xanthine oxidase and 4-hydroxinonenal, as well as a parallel increase in the antioxidant enzymes glutathione peroxidase and superoxide dismutase. Moreover, sulforaphane stimulated the cytoprotective heme oxygenase-1 (HO-1) (38%). Oxidative markers correlated with ERK 1/2 activation. In the MI+SFN group, up-regulation of ERK 1/2 (34%) and Akt (35%), as well as down-regulation of p38 (52%), was observed. This change in the prosurvival kinase balance in the MI+SFN group was related to a down-regulation of apoptosis pathways (Bax/Bcl-2/caspase-3). Sulforaphane was unable to modulate autophagy. Taken together, sulforaphane increased HO-1, which may generate a redox environment in the cardiac tissue favorable to activation of prosurvival and deactivation of prodeath pathways. In conclusion, this natural compound contributes to attenuation of the fibrotic process, which may contribute to mitigation against the progression of cardiac remodeling postinfarction.

Validation of an arterial constitutive model accounting for collagen content and crosslinking

During the progression of pulmonary hypertension (PH), proximal pulmonary arteries (PAs) increase in both thickness and stiffness. Collagen, a component of the extracellular matrix, is mainly responsible for these changes via increased collagen fiber amount (or content) and crosslinking. We sought to differentiate the effects of collagen content and cross-linking on mouse PA mechanical changes using a constitutive model with parameters derived from experiments in which collagen content and cross-linking were decoupled during hypoxic pulmonary hypertension (HPH). We employed an eight-chain orthotropic element model to characterize collagen’s mechanical behavior and an isotropic neo-Hookean form to represent elastin. Our results showed a strong correlation between the material parameter related to collagen content and measured collagen content (R2=0.82, P<0.0001) and a moderate correlation between the material parameter related to collagen crosslinking and measured crosslinking (R2=0.24, P=0.06). There was no significant change in either the material parameter related to elastin or the measured elastin content from histology. The model-predicted pressure at which collagen begins to engage was ∼25mmHg, which is consistent with experimental observations. We conclude that this model may allow us to predict changes in the arterial extracellular matrix from measured mechanical behavior in PH patients, which may provide insight into prognoses and the effects of therapy. Statement of significanceThe literature has proposed several constitutive models to describe the mechanical effects of arterial collagen but none separates collagen content from crosslinking. Given that both are critical to arterial mechanics, the novel model described here does so. Furthermore, our novel model is well tested by experimental data; model parameters were reasonably correlated with measured collagen content and crosslinking and the model-predicted collagen transition stretch was consistent with that obtained experimentally. Given that arterial collagen structural changes and collagen engagement are critical to arterial stiffening in several disease states, this model, by linking mechanical and biological properties, may allow us to predict important biological changes during disease progression from measured mechanical behavior.

Abstract 15999: The Divergent Effects of Exercise Training After Myocardial Infarction or Pressure Overload Depend Critically on Endothelial Nitric Oxide Synthase

AIMS: Beneficial effects of aerobic exercise training are widely recognized. However, previously we discovered that the positive effects of exercise depend on the underlying cause of cardiac failure. Here we tested the hypothesis that endothelial nitric oxide synthase (eNOS) dependent regulation of the balance between nitric oxide and superoxide (O2•-) is critically involved in determining the effects of exercise. METHODS: Mice were exposed to 8 weeks of voluntary wheel running exercise training (EX) or sedentary housing (SED) immediately following myocardial infarction (MI), pressure overload from a transverse aortic constriction (TAC), or sham (SH) surgery. Subsequently, left ventricular (LV) ejection fraction (EF) was measured by echocardiography and Picrosirius Red staining was performed to measure collagen content. Additionally, total and NOS-dependent LV O2•- were measured using lucigenin-enhanced chemiluminescence without or with NOS inhibitor, L-NAME. eNOS uncoupling was evaluated by determining eNOS monomer dimer protein ratio and peroxynitrite (ONOO-) levels were measured through luminol-enhanced chemiluminescence. RESULTS: Cardiac dysfunction and fibrosis were ameliorated by exercise in MI but not in TAC mice (Table 1). MI and TAC both increased LV O2•- levels. Strikingly, EX diminished O2•- generation in MI, but exacerbated O2•- generation in TAC (Table 1). Furthermore, the EX-induced increase in O2•- levels in TAC were largely NOS-dependent. Accordingly, MI and TAC-induced eNOS uncoupling was normalized by EX in MI but aggravated in TAC mice (Table 1). Similarly, increased ONOO- levels following MI and TAC were diminished by EX in MI, but exacerbated by EX in TAC (Table 1). CONCLUSIONS: EX reduces eNOS-mediated cardiac oxidative stress in MI. In contrast, beneficial effects of EX are lacking in cardiac pressure-overload following TAC, due to EX-induced aggravation of ONOO- formation, eNOS uncoupling and concomitant oxidative stress.

Enalaprilat-induced cardiac fibroblast proliferation via regulation of transforming growth factor beta 1 (TGF-β1)-expressing anti-angiotensin II (Ang II)

The objectives of this study were to investigate the effects of the angiotensin I-converting enzyme inhibitor enalaprilat (Ena) on the proliferation, hydroxyproline content, and transforming growth factor β1 (TGF-β1) protein expression of neonatal cardiac fibroblasts and to probe its anti-cardiac fibrosis mechanism. Cardiac fibroblast (CFb) was isolated using the trypsin digestion method. Methyl thiazolyl tetrazolium (MTT) colorimetric assay was performed to evaluate cell proliferation, a hydroxyproline method of determination was used to measure collagen content, and reverse transcription-polymerase chain reaction and flow cytometry were used to detect mRNA TGF-β1 and protein levels, respectively, with Ena. Ena decreased dramatically the MTT value, the hydroxyproline content, and inhibited TGF-β1 transcription and protein expression in neonatal rat CFb. The antiproliferative effects of Ena on CFb may be attributed to the inhibition of TGF-β1 transcription and protein expression in cardiac fibroblasts and the antagonistic action of angiotensin II (Ang II). Key words: Enalaprilat, cardiac fibroblast, hydroxyproline, transforming growth factor-β1.

THE EFFECT OF PREGNANCY AND POSTPARTUM RECOVERY ON THE VISCOELASTIC BEHAVIOR OF THE RAT CERVIX

The objective of this study was to elucidate the normal functional adaptations of the cervix in pregnancy. Utilizing a Long-Evans rodent model, the cervix was divided into distal and proximal portions for virgin, mid-pregnant, and four weeks postpartum animals. The quasi-linear viscoelastic theory describes the elastic and viscous behavior of the cervix. A hydroxyproline assay was used to measure collagen content. The nonlinearity of the elastic response significantly increased throughout the entire cervix during pregnancy when compared to virgin samples (p < 0.05) and was similar to virgin samples postpartum. All viscous behavior, except for the short-term relaxation of the proximal cervix, significantly differed for pregnant specimens (p < 0.05) and remained similar to pregnant samples postpartum. Collagen content was found to increase by mid-pregnancy only in the proximal cervix when compared to virgin. Distal and proximal portions, however, were found to differ in collagen content at all time points (p < 0.05). This study finds that the cervix becomes elastically stiffer with increasing strain and exhibits increased viscous behavior during pregnancy, with incomplete recovery postpartum. These alterations allow for quick dissipation of loads, and are likely related to altered matrix organization and porosity reported by others.

Effects of soybean peptide and collagen peptide on collagen synthesis in normal human dermal fibroblasts

The collagen present in the dermis of the skin is a fibrous protein that fills the gaps between cells and helps maintain tissue flexibility. Effectively increasing the collagen present in the skin is an important goal for cosmetic research. Recent research has shown that soybean peptide (SP) has anti-fatigue activity, antioxidant activity, and the ability to increase type I collagen, while collagen peptide (CP) has the ability to enhance corneal moisture content and viscoelasticity, as well as to increase levels of hyaluronic acid synthesizing enzymes in human skin. Little documented research, however, has been conducted on collagen formation in relation to these peptides. Therefore, this research applied SP and CP with molecular weights primarily around 500 and preparations containing both SP and CP to normal human dermal fibroblasts together with magnesium ascorbyl phosphate (VC-PMg), and used real-time PCR to determine the gene expression of type I collagen (COL1A1), which contributes to collagen synthesis, and Smad7, which contribute to collagen breakdown. In addition, enzyme linked immuno sorbent assay (ELISA) was used to measure collagen content in the media. COL1A1 gene expression at 24 h after sample addition showed higher tendency in all samples and increased with time at 4, 8 and 24 h after addition. Smad7 gene expression was not substantially different at 4 h after addition. matrix metalloproteinase-1 gene expression was higher following SP addition, but was lower after the addition of CP and SP+CP. Medium collagen content was higher in all samples and increased with time at 8 h after addition. Collagen levels were higher when SP and CP were added together.

Shedding Light on Bioabsorbable Stent Struts Seen by Optical Coherence Tomography in the ABSORB Trial

Optical coherence tomography (OCT) is an emerging intracoronary imaging technology that obtains depth-resolved images of light backscattered from the coronary wall with an axial resolution of ≈10 μm. First demonstrated in 2001 in patients,1 intracoronary OCT is now commercially available and is beginning to be adopted in cardiac catheterization laboratories worldwide. An important application of OCT is the investigation of intracoronary stents; ≈100 articles have been published over the past 5 years in which OCT was used for this purpose. OCT is well suited for such studies because (1) its contrast and resolution enable delineation of fine details including device and arterial wall structures; (2) stent healing occurs near the luminal surface, where OCT is most effective; and (3) relative to other imaging modalities, OCT has fewer imaging artifacts that interfere with the appearance of stent struts and surrounding tissues. Article see p 2288 Recently, researchers have found that OCT may also be well suited for investigating the fate of bioabsorbable stents, including bioabsorbable vascular scaffolds (BVS).2,–,9 These new devices are of particular interest because they offer the potential to facilitate coronary revascularization while subsequently being dissolved and assimilated into the artery wall. Resorption in this manner may overcome many of the limitations of bare-metal and drug-eluting stents by leaving behind a revascularized coronary artery without any residual “foreign” material that can promote restenosis or precipitate late stent thrombosis. Given the recent introduction of new generations of bioabsorbable stents, it is critical to understand and monitor …

Monitoring Collagen Transcription by Vascular Smooth Muscle Cells in Fibrin-Based Tissue Constructs

Current methods for measuring collagen content in engineered tissues are incompatible with monitoring of collagen production because they require destruction of the tissue. We have implemented a luciferase-based strategy to monitor collagen production noninvasively. Fibrin-based tissue constructs made using vascular smooth muscle cells stably transfected with a collagen I promoter/luciferase transgene developed with collagen content comparable to control cells, but could be imaged noninvasively to follow collagen transcription during tissue growth in vitro. We showed that these cells reported collagen I production at the transcriptional level in response to the growth factor transforming growth factor-beta1 and fibrinolytic inhibition by epsilon-aminocaproic acid and that these changes were consistent with changes at the mRNA and protein levels. As these cells report collagen changes instantly and without tissue destruction, they will facilitate construct optimization using multiple stimuli to produce functional engineered tissues.

Early cytotoxic effects of ochratoxin A in rat liver: A morphological, biochemical and molecular study

We characterized the overall early effect of chronic ochratoxin A (OTA) treatment on rat liver, analyzing different aspects related to: (i) fibrosis, by measuring collagen content and turnover, and α-smooth muscle actin (αSMA); (ii) oxidative stress and stress response, by analyzing protein carbonylation, superoxide dismutase (SOD) and heat shock protein (HSP70) gene expression; (iii) the possible tumor promoter effect, evaluating cadherin and connexin (CX) mRNA levels. Light microscopy analysis showed no histological differences in OTA-treated and control (CT) rats. Collagen content, determined by computer analysis of Sirius red-stained liver sections, was similar in both groups. In liver homogenates COL-I, COL-III, TIMP-1 and TGF-β1 mRNA levels and αSMA were unaffected by OTA. Matrix metalloproteinase (MMP)-1, MMP-2 and MMP-9 protein levels were also similar in the two groups. Protein carbonylation, a marker of severe oxidative stress, was not evident in the homogenates of OTA-treated livers; superoxide dismutase (SOD) mRNA tended to be lower and HSP70 was strongly down-regulated. OTA reduced E-cadherin and DSC-2 transcription, and down-regulated liver CX26, CX32 and CX43. In conclusion, these in vivo results show that OTA-induced liver injury involves a reduction in the ability to counterbalance oxidative stress, maybe leading to altered gap junction intercellular communication and loss of cell adhesion and polarity. This suggests that mild oxidative damage might be a key factor, in combination with other cytotoxic effects, in triggering the promotion of liver tumors after exposure to OTA.

Clinical review: Healing in gastrointestinal anastomoses, Part I

Gastrointestinal healing is a topic rarely reviewed in the literature, yet it is of paramount importance to the surgeon. Failure of anastomotic healing may lead to life-threatening complications, additional surgical procedures, increased length of stay, increased cost, long-term disability, and reduced quality of life for the patient. The goal of this article is to review the biological response to wounded tissue, to outline discrete differences between skin and gastrointestinal healing, to discuss local and systemic factors important to gastrointestinal healing, and to compare methods of measuring collagen content and strength of the newly formed anastomosis. Part II of this review will focus on techniques and therapies available to optimize anastomotic healing.

Metabolism and composition of the canine anterior cruciate ligament relate to differences in knee joint mechanics and predisposition to ligament rupture

Purpose: The objective of this study was to determine whether differences in the composition and metabolism of the extracellular matrix (ECM) in canine anterior cruciate ligaments (ACLs) might relate to mechanical properties of the canine knee. Variations in ACL biochemistry and knee mechanics could account for divergent predispositions to ligament rupture. Methods: Eleven knee joints were obtained from both cadaveric Labrador Retrievers (rupture predisposed) and ex-racing Greyhounds (non-rupture predisposed). Anterioposterior laxity and tensile testing determined mechanical properties of the knee joints and ACL samples respectively. The thermal properties of the collagenous matrix were determined by differential scanning calorimetry (DSC) and the biochemical properties by measuring collagen content, collagen cross-links, glycosaminoglycan (GAG) levels, matrix metalloproteinase-2 (MMP-2), tissue inhibitors of metalloproteinase (TIMP). Results: The anterioposterior laxity was significantly greater ( p=0.04) in the Labrador Retriever knee joints, and their ACLs tended to be weaker ( p=0.06). Greater collagen turnover was demonstrated by significantly higher ( p=0.02) concentrations of pro-MMP-2, and lower enthalpy of denaturation ( p=0.05) in Labrador Retriever ACLs. Conclusions: The different metabolism of the collagenous matrix in the ACLs of dogs predisposed to rupture was related to greater knee joint laxity and lower ligament material properties (ultimate tensile stress). This may be suggestive of a link between ligament rupture and eventual knee osteoarthritis in both dogs and humans.