Insoluble Collagen Research Articles

Structural comparison of ColH and ColG collagen-binding domains from Clostridium histolyticum.

Clostridium histolyticum secretes collagenases, ColG and ColH, that cause extensive tissue destruction in myonecrosis. The C-terminal collagen-binding domain (CBD) of collagenase is required for insoluble collagen fibril binding and subsequent collagenolysis. The high-resolution crystal structures of ColG-CBD (s3b) and ColH-CBD (s3) are reported in this paper. The new X-ray structure of s3 was solved at 2.0-Å resolution (R = 17.4%; R(free) = 23.3%), while the resolution of the previously determined s3b was extended to 1.4 Å (R = 17.9%; R(free) = 21.0%). Despite sharing only 30% sequence identity, the molecules resemble one another closely (root mean square deviation [RMSD] C(α) = 1.5 Å). All but one residue, whose side chain chelates with Ca(2+), are conserved. The dual Ca(2+) binding site in s3 is completed by an unconserved aspartate. Differential scanning calorimetric measurements showed that s3 gains thermal stability, comparable to s3b, by binding to Ca(2+) (holo T(m) = 94.1°C; apo T(m) = 70.2°C). holo s3 is also stabilized against chemical denaturants urea and guanidine HCl. The three most critical residues for collagen interaction in s3b are conserved in s3. The general shape of the binding pocket is retained by altered loop structures and side chain positions. Small-angle X-ray scattering data revealed that s3 also binds asymmetrically to minicollagen. Besides the calcium-binding sites and the collagen-binding pocket, architecturally important hydrophobic residues and the hydrogen-bonding network around the cis-peptide bond are well conserved within the metallopeptidase subfamily M9B. CBDs were previously shown to bind to the extracellular matrix of various tissues. Compactness and extreme stability in physiological Ca(2+) concentration possibly make both CBDs suitable for targeted growth factor delivery.

Effect of Phosphoric Acid on the Degradation of Human Dentin Matrix

This study determined if dentin proteases are denatured by phosphoric acid (PA) used in etch-and-rinse dentin adhesives. Dentin beams were completely demineralized with EDTA for 30 days. We “acid-etched” experimental groups by exposing the demineralized dentin beams to 1, 10, or 37 mass% PA for 15 sec or 15 min. Control beams were not exposed to PA but were incubated in simulated body fluid for 3 days to assay their total endogenous telopeptidase activity, by their ability to solubilize C-terminal crosslinked telopeptides ICTP and CTX from insoluble dentin collagen. Control beams released 6.1 ± 0.8 ng ICTP and 0.6 ± 0.1 ng CTX/mg dry-wt/3 days. Positive control beams pre-incubated in p-aminophenylmercuric acetate, a compound known to activate proMMPs, released about the same amount of ICTP peptides, but released significantly less CTX. Beams immersed in 1, 10, or 37 mass% PA for 15 sec or 15 min released amounts of ICTP and CTX similar to that released by the controls (p > 0.05). Beams incubated in galardin, an MMP inhibitor, or E-64, a cathepsin inhibitor, blocked most of the release of ICTP and CTX, respectively. It is concluded that PA does not denature endogenous MMP and cathepsin activities of dentin matrices.

Emerging rules for inducing organ regeneration

We review the available evidence for regeneration of adult organs of very diverse nature and examine the applicability of simple rules that can be used to summarize these treatments. In the field of regenerative medicine no widely accepted paradigm is currently available that can guide formulation of new theories on the mechanism of regeneration in adults and open new directions for improved regeneration outcomes. The four rules have emerged from multiyear quantitative studies with skin and peripheral nerve regeneration using scaffold libraries based on a simple, well-defined collagen scaffold. These largely quantitative rules distinguish sharply between spontaneously regenerative and nonregenerative tissues, select the two reactants that are required for regeneration, recognize the essential modification of the wound healing process that must be realized prior to regeneration, and identify three structural features of scaffolds that are required for regenerative activity. The combined evidence points at certain requirements for the structure of a collagen scaffold with regenerative activity. An active scaffold emerges as a temporarily insoluble collagen surface, equipped with sufficient ligands for integrins of contractile cells, that inhibits wound contraction while also serving as a topographic template for new stroma synthesis. The four rules, based on studies with just two organs (skin and peripheral nerves), are now viewed in the context of ongoing studies using scaffolds based on decellularized matrices, which are mostly based on collagen. Decellularized matrices have been used during the past few years to regenerate, in whole or in part, the urethra, the abdominal wall, the Achilles tendon, the bladder, the trachea and other organs in several animal models and occasionally in humans. Although these acellular matrices are distinctly different from simple collagen scaffolds, and the methods used by the investigators are still evolving, the results obtained are shown to be broadly consistent with the predictions of the four rules. Future use or adaptations of these largely quantitative rules could account more satisfactorily for problems, such as imperfect function of regenerated organs, that are currently encountered by researchers. It could also further the explanation of the mechanism of regeneration at the cellular and molecular level.

Collagen Cross-Linking But Not Collagen Amount Associates With Elevated Filling Pressures in Hypertensive Patients With Stage C Heart Failure

We investigated whether the quality of myocardial collagen associates with elevated left-sided filling pressures in 38 hypertensive patients with stage C chronic heart failure. Filling pressures were assessed invasively measuring pulmonary capillary wedge pressure. Left ventricular chamber stiffness constant was calculated from the deceleration time of the early mitral filling wave. The fraction of myocardial volume occupied by total collagen tissue and collagen type I fibers was assessed histomorphologically. The degree of collagen cross-linking (CCL), which determines the formation of insoluble stiff collagen, was assessed by colorimetric and enzymatic procedures. The expression of lysyl oxidase (LOX), which regulates CCL, was assessed by Western blot. Compared with patients with normal pulmonary capillary wedge pressure (≤12 mm Hg; n=16), patients with elevated pulmonary capillary wedge pressure (>12 mm Hg; n=22) exhibited increases of left ventricular chamber stiffness constant, fraction of myocardial volume occupied by total collagen tissue, fraction of myocardial volume occupied by collagen type I fibers, CCL, insoluble stiff collagen, and LOX. Pulmonary capillary wedge pressure was correlated with left ventricular chamber stiffness constant ( r =0.639; P <0.001), insoluble stiff collagen ( r =0.474; P <0.005), CCL ( r =0.625; P <0.001), and LOX ( r =0.410; P <0.05) in all of the patients but not with fraction of myocardial volume occupied by total collagen tissue or fraction of myocardial volume occupied by collagen type I fibers. In addition, CCL was correlated with insoluble stiff collagen ( r =0.612; P <0.005), LOX ( r =0.538; P <0.01), left ventricular chamber stiffness constant ( r =0.535; P <0.005), peak filling rate ( r =−0.343; P <0.05), ejection fraction ( r =−0.430; P <0.01), and amino-terminal propeptide of brain natriuretic peptide ( r =0.421; P <0.05) in all of the patients. These associations were independent of confounding factors. These findings indicate that, in hypertensive patients with stage C heart failure, it is only the quality of collagen (ie, degree of cross-linking) that associates with elevated filling pressures. It is suggested that LOX-mediated excessive CCL facilitates the increase in left ventricular stiffness with the resulting elevation of filling pressures in these patients.

Time course of right ventricular pressure-overload induced myocardial fibrosis: relationship to changes in fibroblast postsynthetic procollagen processing

Myocardial fibrillar collagen is considered an important determinant of increased ventricular stiffness in pressure-overload (PO)-induced cardiac hypertrophy. Chronic PO was created in feline right ventricles (RV) by pulmonary artery banding (PAB) to define the time course of changes in fibrillar collagen content after PO using a nonrodent model and to determine whether this time course was dependent on changes in fibroblast function. Total, soluble, and insoluble collagen (hydroxyproline), collagen volume fraction (CVF), and RV end-diastolic pressure were assessed 2 days and 1, 2, 4, and 10 wk following PAB. Fibroblast function was assessed by quantitating the product of postsynthetic processing, insoluble collagen, and levels of SPARC (secreted protein acidic and rich in cysteine), a protein that affects procollagen processing. RV hypertrophic growth was complete 2 wk after PAB. Changes in RV collagen content did not follow the same time course. Two weeks after PAB, there were elevations in total collagen (control RV: 8.84 ± 1.03 mg/g vs. 2-wk PAB: 11.50 ± 0.78 mg/g); however, increased insoluble fibrillar collagen, as measured by CVF, was not detected until 4 wk after PAB (control RV CVF: 1.39 ± 0.25% vs. 4-wk PAB: 4.18 ± 0.87%). RV end-diastolic pressure was unchanged at 2 wk, but increased until 4 wk after PAB. RV fibroblasts isolated after 2-wk PAB had no changes in either insoluble collagen or SPARC expression; however, increases in insoluble collagen and in levels of SPARC were detected in RV fibroblasts from 4-wk PAB. Therefore, the time course of PO-induced RV hypertrophy differs significantly from myocardial fibrosis and diastolic dysfunction. These temporal differences appear dependent on changes in fibroblast function.

Effects of the absence of procollagen C-endopeptidase enhancer-2 on myocardial collagen accumulation in chronic pressure overload

Cardiac interstitial fibrillar collagen accumulation, such as that associated with chronic pressure overload (PO), has been shown to impair left ventricular diastolic function. Therefore, insight into cellular mechanisms that mediate excessive collagen deposition in the myocardium is pivotal to this important area of research. Collagen is secreted as a soluble procollagen molecule with NH(2)- and COOH (C)-terminal propeptides. Cleavage of these propeptides is required for collagen incorporation to insoluble collagen fibrils. The C-procollagen proteinase, bone morphogenic protein 1, cleaves the C-propeptide of procollagen. Procollagen C-endopeptidase enhancer (PCOLCE) 2, an enhancer of bone morphogenic protein-1 activity in vitro, is expressed at high levels in the myocardium. However, whether the absence of PCOLCE2 affects collagen content at baseline or after PO induced by transverse aortic constriction (TAC) has never been examined. Accordingly, in vivo procollagen processing and deposition were examined in wild-type (WT) and PCOLCE2-null mice. No significant differences in collagen content or myocardial stiffness were detected in non-TAC (control) PCOLCE2-null versus WT mice. After TAC-induced PO, PCOLCE2-null hearts demonstrated a lesser collagen content (PCOLCE2-null TAC collagen volume fraction, 0.41% ± 0.07 vs. WT TAC, 1.2% ± 0.3) and lower muscle stiffness compared with WT PO hearts [PCOLCE2-null myocardial stiffness (β), 0.041 ± 0.002 vs. WT myocardial stiffness, 0.065 ± 0.001]. In addition, in vitro, PCOLCE2-null cardiac fibroblasts exhibited reductions in efficiency of C-propeptide cleavage, as demonstrated by increases in procollagen α1(I) and decreased levels of processed collagen α1(I) versus WT cardiac fibroblasts. Hence, PCOLCE2 is required for efficient procollagen processing and deposition of fibrillar collagen in the PO myocardium. These results support a critical role for procollagen processing in the regulation of collagen deposition in the heart.

Age‐related expression, enzymatic solubility and modification with advanced glycation end‐products of fibrillar collagens in mouse lung

Changes in the expression of fibrillar collagens and post-translational modifications with advanced glycation end-products (AGEs) are often associated with tissue aging. Less is known about age-related changes in mouse lung tissue. Therefore, we studied the expression level and AGE load of fibrillar collagens in lungs from young (≤6months), adult (15months) and old (≥25months) mice. The mRNA expression level was reduced in adult and old mice compared with the young. Old mice also showed a reduced protein level, whereas the adults even had more collagen protein. Fractionating of the fibrillar collagens into enzyme-soluble and insoluble collagens revealed a reduced solubility of collagens in old age. The enzymatic solubility of fibrillar collagens correlated inversely with the AGE load in the insoluble collagen as detected by the AGE-related fluorescence. While the intensity of the AGE-related fluorescence was increased in fibrillar collagens in response to age, the fluorescing AGE variant argpyrimidine was less affected. In summary, aging causes a reduced expression, lower enzymatic solubility and increased AGE load of fibrillar collagens in mouse lung tissue, but not all changes occur gradually with age.

Rap 1a small GTPase alters extracellular matrix (ECM) deposition in response to TGF‐β1

Cardiac fibroblasts are essential to cardiac remodeling by regulating ECM production, maturation and degradation. TGF‐β, transforming growth factor beta, can regulate fibroblast differentiation and activation, and therefore tissue fibrosis. Rap1a, a Ras‐like GTPase, has been implicated in cell migration and attachment, however, its role in regulation of ECM has yet to be determined. In this study, we examined the impact of Rap1 deficiency on cardiac ECM remodeling.Analysis of left ventricular tissue sections from Rap1a deficient heterozygous or knockout (HET or KO) mice revealed significant decreases in total (picrosirius red‐PASR), soluble and insoluble collagen when compared to wild‐type (WT) controls (Table I). Isolated cardiac fibroblasts from heterozygous Rap1a mice showed a blunted collagen response to TGF‐β, suggesting an alteration in the normal pro‐fibrotic response to this cytokine. Genetic ablation of Rap1a shows an impact on regulation of ECM which may lead to alterations in cardiac remodeling and hypertrophic response.

Collagen deposition and cellular viability among UVB irradiated human dermal fibroblasts treated by platelets

Prolonged or repeated low energy ultraviolet B (UVB) irradiation that can inhibit fibroblast proliferation, collagen synthesis, and stimulate collagen degradation is basic concept of skin photo-aging. Recently, a study showed that human skin fibroblasts treated with platelets resulted in a significant higher proliferation rates compared to placebo. The effect of platelets in photo aged human skin fibroblasts, however, is still unclear. To know the effect of platelets to UVB irradiated fibroblasts focusing on proliferation rate and extracellular collagen deposition, various platelet concentrations were given to UVB irradiated normal human dermal fibroblasts. Fibroblast viability rates were determined by MTT-assay, whereas collagen deposition levels were determined by insoluble collagen Sirius red-assay. Data were analyzed by Kruskal-Wallis H and post-hoc by Mann-Whitney U. p <0.05 is accepted for significance difference. A group treated by 1.25 normal platelet concentration showed an increase cellular viability rates and collagen deposition levelsignificantly (p<0.01). As a conclusion, platelet in 1.25 normal platelet concentrations may be a promising modality for the treatment of photo-aging skin. Key words: Platelet, ultraviolet B (UVB), fibroblast proliferation, collagen deposition.

Analysis of advanced glycation end product modifications in collagen of cardiac tissue and graft material from patients suffering from coronary heart disease

Background: Advanced glycation end products (AGEs) seem to be involved in ageing as well as in the development of cardiovascular diseases. During ageing, the AGEs accumulate in matrix proteins like collagen and fibronectin inducing tissue stiffness. The Bypass operation is the most common intervention in patients suffering from coronary heart disease. However as the majority of the patients are older than 60, the graft material (vein or artery) of these patients is probably enriched on AGE-modifications. To which extent these modifications may affect the quality and functionality of bypass graft material and the heart function after operation is unknown. We analyzed the AGE-modifications in the collagen from the right atrium and residual bypass graft material (saphenous vein and internal thoracic artery) to elucidate in which extension the collagen of graft material is AGE-modified. Methods: Collagen type I and III (soluble fraction) were isolated from veins, arteries and the right atrium by proteolysis with pepsin. The residual insoluble fraction was extracted by digestion with collagenase type I and proteinase K. Collagen was quantified by 4-hydroxyproline assay and AGEs by the AGE intrinsic fluorescence (360nm excitation and 440nm emission). Results: The AGE fluorescence in patient material (vein, atrium) increased with patients age (p=0.0025). Decreasing amounts of extracted insoluble collagen correlate with increased AGE fluorescence (p=0.0001). Comparing vein, artery and atrium of the same patients we isolated equal levels of soluble and insoluble collagen in arteries and atriums, whereas only in veins increased amounts of soluble collagen were extracted in comparison to the insoluble fraction (p<0.05). The soluble fraction in every tissue was significantly less modified in comparison to the insoluble collagen (p<0.05). Conclusion: Ageing is reflected by an increase in AGE related tissue collagen fluorescence. Increase crosslinking of collagen, reflected by high AGE fluorescence, may be the cause of reduced amounts of insoluble extracted collagen. High levels of AGE-modifications are known to block the proteolytic degradation by matrix metalloproteinase. Arteries and cardiac tissue (right atrium) showed significantly more modifications compared to vein graft material of the same patients. Our results may indicate why veins and arteries behave differently as bypass graft material. This will be further evaluated from our group.

Effects of Prolonged Stretching and Thermotherapy on Muscle Contracture of Immobilized Rat Soleus Muscle

Purpose) We examined the effects of prolonged stretching and thermotherapy on muscle contracture of immobilized rat soleus muscles by determining ankle joint range of motion (ROM), collagen fibril arrangement in the endomysium and insoluble collagen content. (Methods) In the experimental group, bilateral ankle joints of each rat were fixed in full plantar flexion with plaster casts for four weeks. Then, the experimental group animals were randomly partitioned into five groups: 1) immobilization alone; and 2) spontaneous recovery, 3) prolonged stretching, 4) thermotherapy, and 5) prolonged stretching immediately following thermotherapy for 2 weeks after cast removal. Prolonged stretching of the soleus muscle (30 min/day) was conducted by maintaining the dorsiflexion position of the ankle joint. Thermotherapy consisted of immersion of bilateral hindlimbs (20 min/day) in hot water (42 °C). Under anesthesia, both treatments were performed six days per week during the two-week remobilization. (Results) Immobilization led to decreased ROM, collagen fibril movement in the endomysium and increased in - soluble collagen. Prolonged stretching and the combination of stretching and thermotherapy accelerated ameliora- tion of the ROM limitation and diminished collagen fibril movement. (Conclusion) Our findings suggest that while thermotherapy does not exert a specific effect on muscle contracture, prolonged stretching can promote recovery.

Advanced glycation end-products as markers of aging and longevity in the long-lived Ansell's mole-rat (Fukomys anselli).

Mole-rat of the genus Fukomys are mammals whose life span is strongly influenced by reproductive status with breeders far outliving nonbreeders. This raises the important question of whether increased longevity of the breeders is reflected in atypical expression of biochemical markers of aging. Here, we measured markers of glycation and advanced glycation end-products formed in insoluble skin collagen of Ansell's mole-rat Fukomys anselli as a function of age and breeding status. Glucosepane, pentosidine, and total advanced glycation end-product content significantly increased with age after correction for breeder status and sex. Unexpectedly, total advanced glycation end-products, glucosepane, and carboxymethyl-lysine (CML) were significantly higher in breeders versus nonbreeders suggesting that breeders have evolved powerful defenses against combined oxidant and carbonyl stress compared with nonbreeders. Most interestingly, when compared with other mammals, pentosidine formation rate was lower in mole-rat compared with other short-lived rodents confirming previous observations of an inverse relationship between longevity and pentosidine formation rates in skin collagen.

Novel Application of Human Periodontal Ligament Stem Cells and Water-Soluble Chitin for Collagen Tissue Regeneration:In VitroandIn VivoInvestigations

Human periodontal ligament stem cells (hPDLSCs) have been proposed as an alternative to conventional cosmetic fillers because they display an innate ability to synthesize collagen. The aims of this study were to determine the effects of water-soluble chitin (WSC) on the proliferation and migration of hPDLSCs, and to quantify collagen synthesis in vitro and in vivo compared with human adipose-derived stem cell (hADSC)s. hPDLSCs were isolated from healthy extracted teeth, and the cell proliferation and cell migration capacities of untreated hPDLSCs (control group) and WSC-treated hPDLSCs (test group) were compared. Insoluble/soluble collagen synthesis were also assessed, and collagen related markers were evaluated including lysyl oxidase (LOX), lysyl oxidase like (LOXL)1, LOXL2, and hydroxyproline. In vivo collagen formation was examined by transplanting hyaluronic acid as a cell carrier into the subcutaneous pockets of immunocompromised mice in the control and test groups; histology and immunohistochemistry analyses were performed 4 (n=4) and 8 (n=4) weeks later. There was a dose-dependent enhancement of hPDLSCs proliferation in the test group, and a concomitant reduction in cell migration. The amount of insoluble collagen formed was greater in the test group than in the control group (p<0.05), whereas soluble collagen formation was significantly reduced in the test group (p<0.05). The histology and immunohistochemistry results revealed that the amount of collagen formed in vivo was greater in WSC-treated hPDLSCs than in the control cells at 4 and 8 weeks (p<0.05), and histometric analysis at 8 weeks revealed that enhancement of collagen formation by hPDLSCs was greater than by hADSCs. These results indicate that WSC modulates the properties of hPDLSCs, rendering them more suitable for cosmetic soft-tissue augmentation.

Residual transglutaminase in collagen – Effects, detection, quantification, and removal

In the present study, we developed an enzyme-linked immunosorbent assay (ELISA) for microbial transglutaminase (mTG) from Streptomyces mobaraensis to overcome the lack of a quantification method for mTG. We further performed a detailed follow-on-analysis of insoluble porcine collagen type I enzymatically modified with mTG primarily focusing on residuals of mTG. Repeated washing (4×) reduced mTG-levels in the washing fluids but did not quantitatively remove mTG from the material (p<0.000001). Substantial amounts of up to 40% of the enzyme utilized in the crosslinking mixture remained associated with the modified collagen. Binding was non-covalent as could be demonstrated by Western blot analysis. Acidic and alkaline dialysis of mTG treated collagen material enabled complete removal the enzyme. Treatment with guanidinium chloride, urea, or sodium chloride was less effective in reducing the mTG content.

Obstruction-Induced Pulmonary Vascular Remodeling

Pulmonary obstruction occurs in many common forms of congenital heart disease. In this study, pulmonary artery (PA) banding is used as a model for pulmonary stenosis. Significant remodeling of the vascular bed occurs as a result of a prolonged narrowing of the PAs, and here we quantify the biophysical and molecular changes proximal and distal to the obstruction. Main and branch PAs are harvested from banded and sham rabbits and their mechanical properties are assessed using a biaxial tensile tester. Measurements defined as initial and stiff slopes are taken, assuming a linear region at the start and end of the J-shaped stress-strain curves, along with a transitional knee point. Collagen, elastin assays, Movat's pentachrome staining, and Doppler protocols are used to quantify biochemical, structural, and physiological differences. The banded main PAs have significantly greater initial slopes while banded branch PAs have lower initial slopes; however, this change in mechanical behavior cannot be explained by the assay results as the elastin content in both main and branch PAs is not significantly different. The stiff slopes of the banded main PAs are higher, which is attributed to the significantly greater amounts of insoluble collagen. Shifting of the knee points reveals a decreased toe region in the main PAs but an opposite trend in the branch PAs. The histology results show a loss of integrity of the media, increase in ground substance, and dispersion of collagen in the banded tissue samples. This indicates other structural changes could have led to the mechanical differences in banded and normal tissue.

Potential of a custom-designed fluorescence imager combined with multivariate statistics for the study of chemical and mechanical characteristics of beef meat

The potential of fluorescence imaging to discriminate different bovine muscles in relation with animal age, muscle type, chemical and mechanical properties was examined. Twenty-four muscles of three types ( Gluteus medius, Longissimus dorsi, and Semitendinosus) and two animal age groups (10–13-years old and 12–24-months old) were obtained from the carcasses of Limousin breed cows. One hundred and forty-four images were collected at three illuminating conditions (exc 320 nm, exc 380 nm, and white light) using a custom-designed imager. The image cubes were processed using “regionprops” algorithm developed earlier in order to extract image shape features ( number of shapes, area, major- axis- length, eccentricity, and solidity). Extracted image shape features were processed using custom-designed programs. The results of the PLSDA performed on image shape features showed 100% good discrimination for the three types of muscles. Muscle samples were also subjected to chemical analysis (dry matter, fat, pyridinoline, total, insoluble and soluble collagen) and mechanical tests (shear stress and breaking energy). PLSR models indicated relations between extracted image shape features and mechanical properties, i.e., R 2 = 0.69 and RMSEV = 0.514 were observed for breaking energy for adult-animal muscles. Regarding chemical composition, image shape features allowed to predict total collagen of L. dorsi with R 2 = 0.61 and RMSEV = 0.756. This study has demonstrated a promising potential of the custom-designed fluorescence imager combined with multivariate statistical tools in the study of beef meat.

Caveolin-1 is a negative regulator of MMP-1 gene expression in human dermal fibroblasts via inhibition of Erk1/2/Ets1 signaling pathway

Caveolar raft domains, also termed caveolae, are flask shaped invaginations that require the expression of the structural protein caveolin-1 (cav-1). Matrix metalloproteinase 1 (MMP-1) is a collagenase capable of degrading insoluble triple helical collagens. Deregulation of MMP-1 contributes to various pathological processes, including tissue fibrosis and impaired wound healing. In this study we investigated the role of cav-1 in MMP-1 gene regulation in human dermal fibroblasts. Fibroblasts were isolated from healthy subjects. Western blot was used to analyze protein levels and quantitative real time RT-PCR was used to measure mRNA expression. Cells were transiently transfected with siRNA oligos against acid sphingomyelinase (ASMase) and cav-1, or transduced with adenoviruses overexpressing ASMase and cav-1. The specific pharmacological inhibitors UO126 and SP600125 were used to block Erk1/2 and JNK activity. This study shows that siRNA-mediated depletion of ASMase or cav-1, results in upregulation of MMP-1 gene expression. Similarly, MMP-1 expression was decreased after overexpresssion of cav-1 via an adenoviral vector. Depletion of cav-1 had no effect on JNK phosphorylation, while it resulted in an increase in Erk1/2 and Ets1 phosphorylation levels. Furthermore, in cav-1 depleted cells treated with the Erk inhibitor UO126, there was no increase in the levels of phospho-Erk1/2, phospho-Ets1, and MMP-1, suggesting that cav-1 mediated effects on MMP-1 and phospho-Ets1 are Erk1/2 dependent. In conclusion, this study has revealed an important role for cav-1 as a negative regulator of MMP-1 gene expression via inhibition of Erk1/2/Ets1 signaling. Cav-1 could potentially be a therapeutic target in diseases with deregulated extracellular matrix (ECM) turnover.

Vascular tissue construction on poly(ε-caprolactone) scaffolds by dynamic endothelial cell seeding: effect of pore size

In vitro tissue engineering for fabrication of small diameter vascular grafts probably undergoes a sequence of events similar to the in vivo angiogenesis process. In both cases endothelial cells (ECs) play the crucial role in generating a non-thrombogenic vessel lumen and stabilization of ECs in the lumen of new vessels requires the deposition of collagen IV and elastin. Shear stress is an important in vivo signal for inducing synthesis of extracellular matrix (ECM) components, collagen IV and elastin, which form the basement membrane in the case of new blood vessels. Stimulation of ECs may therefore produce collagen and elastin in the lumen of a polymeric scaffold during the vascular tissue-engineering process if appropriate biochemical and mechanical signals are presented. However, the morphology and physicochemical characteristics of polymer scaffolds may also be crucial for EC monolayer formation and ECM deposition. In this study, tubular scaffolds made of biodegradable poly(ε-caprolactone) (PCL) with biomimetic fibrin-based coating were evaluated to compare the effects of pore sizes on surface coverage of ECs and synthesis of ECM under dynamic culture conditions. Actin was stained for identification of cells, while specific antibodies were used for locating collagen IV and elastin deposition on the scaffolds. It was found that dynamic seeding of ECs in the lumen stabilized the cells and aligned them along the direction of flow, with better deposition of insoluble elastin and collagen IV when ∼75% of pores were < 24 µm in diameter. In addition, monolayer on the ε-PCL scaffolds with lower pore sizes was found to produce nitric oxide (NO), indicating a non-thrombogenic EC layer in the lumen.

The Non-phagocytic Route of Collagen Uptake: A DISTINCT DEGRADATION PATHWAY

The degradation of collagens, the most abundant proteins of the extracellular matrix, is involved in numerous physiological and pathological conditions including cancer invasion. An important turnover pathway involves cellular internalization and degradation of large, soluble collagen fragments, generated by initial cleavage of the insoluble collagen fibers. We have previously observed that in primary mouse fibroblasts, this endocytosis of collagen fragments is dependent on the receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180. Others have identified additional mechanisms of collagen uptake, with different associated receptors, in other cell types. These receptors include β1-integrins, being responsible for collagen phagocytosis, and the mannose receptor. We have now utilized a newly developed monoclonal antibody against uPARAP/Endo180, which down-regulates the receptor protein level on treated cells, to examine the role of uPARAP/Endo180 as a mediator of collagen internalization by a wide range of cultured cell types. With the exception of macrophages, all cells that proved capable of efficient collagen internalization were of mesenchymal origin and all of these utilized uPARAP/Endo180 for their collagen uptake process. Macrophages internalized collagen in a process mediated by the mannose receptor, a protein belonging to the same protein family as uPARAP/Endo180. β1-Integrins were found not to be involved in the endocytosis of soluble collagen, irrespectively of whether this was mediated by uPARAP/Endo180 or the mannose receptor. This further distinguishes these pathways from the phagocytic uptake of particulate collagen.

SPARC regulates collagen interaction with cardiac fibroblast cell surfaces

Cardiac tissue from mice that do not express secreted protein acidic and rich in cysteine (SPARC) have reduced amounts of insoluble collagen content at baseline and in response to pressure overload hypertrophy compared with wild-type (WT) mice. However, the cellular mechanism by which SPARC affects myocardial collagen is not clearly defined. Although expression of SPARC by cardiac myocytes has been detected in vitro, immunohistochemistry of hearts demonstrated SPARC staining primarily associated with interstitial fibroblastic cells. Primary cardiac fibroblasts isolated from SPARC-null and WT mice were assayed for collagen I synthesis by [(3)H]proline incorporation into procollagen and by immunoblot analysis of procollagen processing. Bacterial collagenase was used to discern intracellular from extracellular forms of collagen I. Increased amounts of collagen I were found associated with SPARC-null versus WT cells, and the proportion of total collagen I detected on SPARC-null fibroblasts without propeptides [collagen-α(1)(I)] was higher than in WT cells. In addition, the amount of total collagen sensitive to collagenase digestion (extracellular) was greater in SPARC-null cells than in WT cells, indicating an increase in cell surface-associated collagen in the absence of SPARC. Furthermore, higher levels of collagen type V, a fibrillar collagen implicated in collagen fibril initiation, were found in SPARC-null fibroblasts. The absence of SPARC did not result in significant differences in proliferation or in decreased production of procollagen I by cardiac fibroblasts. We conclude that SPARC regulates collagen in the heart by modulating procollagen processing and interactions with fibroblast cell surfaces. These results are consistent with decreased levels of interstitial collagen in the hearts of SPARC-null mice being due primarily to inefficient collagen deposition into the extracellular matrix rather than to differences in collagen production.