Fibrosis and expression of extracellular matrix proteins in human interventricular septum in aortic valve stenosis and regurgitation.

Valvular heart disease leads to ventricular pressure and/or volume overload. Pressure overload leads to fibrosis, which might regress with its resolution, but the limits and details of this reverse remodeling are not known. To gain more insight into the extent and nature of cardiac fibrosis in valve disease, we analyzed needle biopsies taken from the interventricular septum of patients undergoing surgery for valve replacement focusing on the expression and distribution of major extracellular matrix protein involved in this process. Proteomic analysis performed using mass spectrometry revealed an excellent correlation between the expression of collagen type I and III, but there was little correlation with the immunohistochemical staining performed on sister sections, which included antibodies against collagen I, III, fibronectin, sarcomeric actin, and histochemistry for wheat germ agglutinin. Surprisingly, the immunofluorescence intensity did not correlate significantly with the gold standard for fibrosis quantification, which was performed using Picrosirius Red (PSR) staining, unless multiplexed on the same tissue section. There was also little correlation between the immunohistochemical markers and pressure gradient severity. It appears that at least in humans, the immunohistochemical pattern of fibrosis is not clearly correlated with standard Picrosirius Red staining on sister sections or quantitative proteomic data, possibly due to tissue heterogeneity at microscale, comorbidities, or other patient-specific factors. For precise correlation of different types of staining, multiplexing on the same section is the best approach.

Adipose-Derived Stem Cells in Reinforced Collagen Gel: A Comparison between Two Approaches to Differentiation towards Smooth Muscle Cells.

Scaffolds made of degradable polymers, such as collagen, polyesters or polysaccharides, are promising matrices for fabrication of bioartificial vascular grafts or patches. In this study, collagen isolated from porcine skin was processed into a gel, reinforced with collagen particles and with incorporated adipose tissue-derived stem cells (ASCs). The cell-material constructs were then incubated in a DMEM medium with 2% of FS (DMEM_part), with added polyvinylalcohol nanofibers (PVA_part sample), and for ASCs differentiation towards smooth muscle cells (SMCs), the medium was supplemented either with human platelet lysate released from PVA nanofibers (PVA_PL_part) or with TGF-ß1 + BMP-4 (TGF + BMP_part). The constructs were further endothelialised with human umbilical vein endothelial cells (ECs). The immunofluorescence staining of alpha-actin and calponin, and von Willebrand factor, was performed. The proteins involved in cell differentiation, the extracellular matrix (ECM) proteins, and ECM remodelling proteins were evaluated by mass spectrometry on day 12 of culture. Mechanical properties of the gels with ASCs were measured via an unconfined compression test on day 5. Gels evinced limited planar shrinkage, but it was higher in endothelialised TGF + BMP_part gel. Both PVA_PL_part samples and TGF + BMP_part samples supported ASC growth and differentiation towards SMCs, but only PVA_PL_part supported homogeneous endothelialisation. Young modulus of elasticity increased in all samples compared to day 0, and PVA_PL_part gel evinced a slightly higher ratio of elastic energy. The results suggest that PVA_PL_part collagen construct has the highest potential to remodel into a functional vascular wall.

Minoxidil decreases collagen I deposition and tissue-like contraction in clubfoot-derived cells: a way to improve conservative treatment of relapsed clubfoot?

AIM: Clubfoot is a congenital deformity affecting the musculoskeletal system, resulting in contracted and stiff tissue in the medial part of the foot. Minoxidil (MXD) has an inhibitory effect on lysyl hydroxylase, which influences the quality of extracellular matrix crosslinking, and could therefore be used to reduce the stiffness and to improve the flexibility of the tissue. We assessed the in vitro antifibrotic effects of minoxidil on clubfoot-derived cells. METHODS: Cell viability and proliferation were quantified by xCELLigence, MTS, and LIVE/DEAD assays. The amount of collagen I deposited into the extracellular matrix was quantified using immunofluorescence with subsequent image segmentation analysis, hydroxyproline assay, and Second Harmonic Generation imaging. Extracellular matrix contraction was studied in a 3D model of cell-populated collagen gel lattices. RESULTS: MXD concentrations of 0.25, 0.5, and 0.75 mM inhibited the cell proliferation in a concentration-dependent manner without causing a cytotoxic effect. Exposure to ≥0.5 mM MXD resulted in a decrease in collagen type I accumulation after 8 and 21 days in culture. Changes in collagen fiber assembly were observed by immunofluorescence microscopy and nonlinear optical microscopy (second harmonic generation). MXD also inhibited the contraction of cell-populated collagen lattices (0.5 mM by 22%; 0.75 mM by 28%). CONCLUSIONS: Minoxidil exerts an in vitro inhibitory effect on the cell proliferation, collagen accumulation, and extracellular matrix contraction processes that are associated with clubfoot fibrosis. This study provides important preliminary results demonstrating the potential relevance of MXD for adjuvant pharmacological therapy in standard treatment of relapsed clubfoot.

Increased Collagen Crosslinking in Stiff Clubfoot Tissue: Implications for the Improvement of Therapeutic Strategies.

Congenital clubfoot is a complex musculoskeletal deformity, in which a stiff, contracted tissue forms in the medial part of the foot. Fibrotic changes are associated with increased collagen deposition and lysyl oxidase (LOX)-mediated crosslinking, which impair collagen degradation and increase the tissue stiffness. First, we studied collagen deposition, as well as the expression of collagen and the amount of pyridinoline and deoxypyridinoline crosslinks in the tissue of relapsed clubfoot by immunohistochemistry, real-time PCR, and enzyme-linked immunosorbent assay (ELISA). We then isolated fibroblast-like cells from the contracted tissue to study the potential inhibition of these processes in vitro. We assessed the effects of a LOX inhibitor, ß-aminopropionitrile (BAPN), on the cells by a hydroxyproline assay, ELISA, and Second Harmonic Generation imaging. We also evaluated the cell-mediated contraction of extracellular matrix in 3D cell-populated collagen gels. For the first time, we have confirmed significantly increased crosslinking and excessive collagen type I deposition in the clubfoot-contracted tissue. We successfully reduced these processes in vitro in a dose-dependent manner with 10-40 µg/mL of BAPN, and we observed an increasing trend in the inhibition of the cell-mediated contraction of collagen gels. The in vitro inhibitory effects indicate that BAPN has good potential for the treatment of relapsed and resistant clubfeet.

Increased Microvessel and Arteriole Density in the Contracted Side of the Relapsed Clubfoot.

BACKGROUND: Clubfoot deformity (pes equinovarus) is one of the most common birth defects, and its etiology is still unknown. Initial clubfoot treatment is based on the Ponseti method throughout most of the world. Despite the effectiveness of this therapy, clubfoot may relapse. Recent studies confirm the theory of active fibrotic remodeling processes in the extracellular matrix of the affected tissue. The aim of this study was to clarify whether relapses in clubfoot therapy are associated with altered angiogenesis and to suggest possible regulatory pathways of this pathologic process. METHODS: We compared microvessel density, arteriole density, and concentration of angioproliferative-related proteins found between tissues in the contracted, that is, the medial side (M-side), and noncontracted, that is, the lateral side (L-side) of the relapsed clubfeet. Tissue samples from 10 patients were analyzed. Histopathologic analysis consisted of immunohistochemistry and image analysis. Real-time polymerase chain reaction was used to study mRNA expression. RESULTS: An increase in microvessel and arteriole density was noted in contracted, relapsed clubfoot tissue. This was accompanied by a significant increase in the levels of the vascular endothelial growth factor, vascular endothelial growth factor receptor 2, ß catenin and active ß catenin. Vascular endothelial growth factor, vascular endothelial growth factor receptor 2, and CD31 overexpression was also seen with mRNA analysis. CONCLUSIONS: Increased microvessel and arteriole density in the contracted side of the relapsed clubfoot was noted. These processes are mediated by specific proangiogenic proteins that are overexpressed in the contracted tissue. These findings contribute to the etiology and the development of relapses in the treatment of clubfoot. LEVEL OF EVIDENCE: Level II-analytical and prospective.

Endocardial Fibroelastosis is Secondary to Hemodynamic Alterations in the Chick Embryonic Model of Hypoplastic Left Heart Syndrome.

BACKGROUND: Endocardial fibroelastosis (EFE) is a diffuse thickening of the ventricular endocardium, causing myocardial dysfunction and presenting as unexplained heart failure in infants and children. One of the postulated causes is persistent and increased wall tension in the ventricles. RESULTS: To examine whether reduced ventricular pressure in a chick model of hypoplastic left heart syndrome (HLHS) induced by left atrial ligation (LAL) at embryonic day (ED) 4 is associated with EFE at later stages, myocardial fibrosis was evaluated by histology and immunoconfocal microscopy and mass spectrometry (MS) at ED12. Immunohistochemistry with collagen I antibody clearly showed a significant thickening of the layer of subendocardial fibrous tissue in LAL hearts, and MS proved this significant increase of collagen I. To provide further insight into pathogenesis of this increased fibroproduction, hypoxyprobe staining revealed an increased extent of hypoxic regions, normally limited to the interventricular septum, in the ventricular myocardium of LAL hearts at ED8. CONCLUSIONS: Abnormal hemodynamic loading during heart development leads to myocardial hypoxia, stimulating collagen production in the subendocardium. Therefore, EFE in this chick embryonic model of HLHS appears to be a secondary effect of abnormal hemodynamics. Developmental Dynamics 247:509-520, 2018. © 2017 Wiley Periodicals, Inc.

Proteomic analysis of cardiac ventricles: baso-apical differences.

The heart is characterized by a remarkable degree of heterogeneity. Since different cardiac pathologies affect different cardiac regions, it is important to understand molecular mechanisms by which these parts respond to pathological stimuli. In addition to already described left ventricular (LV)/right ventricular (RV) and transmural differences, possible baso-apical heterogeneity has to be taken into consideration. The aim of our study has been, therefore, to compare proteomes in the apical and basal parts of the rat RV and LV. Two-dimensional electrophoresis was used for the proteomic analysis. The major result of this study has revealed for the first time significant baso-apical differences in concentration of several proteins, both in the LV and RV. As far as the LV is concerned, five proteins had higher concentration in the apical compared to basal part of the ventricle. Three of them are mitochondrial and belong to the "metabolism and energy pathways" (myofibrillar creatine kinase M-type, L-lactate dehydrogenase, dihydrolipoamide dehydrogenase). Myosin light chain 3 is a contractile protein and HSP60 belongs to heat shock proteins. In the RV, higher concentration in the apical part was observed in two mitochondrial proteins (creatine kinase S-type and proton pumping NADH:ubiquinone oxidoreductase). The described changes were more pronounced in the LV, which is subjected to higher workload. However, in both chambers was the concentration of proteins markedly higher in the apical than that in basal part, which corresponds to the higher energetic demand and contractile activity of these segments of both ventricles.

Proteomic analysis of the extracellular matrix in idiopathic pes equinovarus.

Idiopathic pes equinovarus is a congenital deformity of the foot and lower leg defined as a fixation of the foot in adduction, supination, and varus. Although the pathogenesis of clubfoot remains unclear, it has been suggested that fibroblasts and growth factors are involved. To directly analyze the protein composition of the extracellular matrix in contracted tissue of patients with clubfoot. A total of 13 infants with idiopathic clubfoot treated with the Ponseti method were included in the present study. Tissue samples were obtained from patients undergoing surgery for relapsed clubfeet. Contracted tissues were obtained from the medial aspect of the talonavicular joint. Protein was extracted after digestion and delipidation using zip-tip C18. Individual collagenous fractions were detected using a chemiluminescent assay. Amino acid analysis of tissue samples revealed a predominance of collagens, namely collagen types I, III, and VI. The high content of glycine and h-proline suggests a predominance of collagens I and III. A total of 19 extracellular matrix proteins were identified. The major result of the present study was the observation that the extracellular matrix in clubfoot is composed of an additional 16 proteins, including collagens V, VI, and XII, as well as the previously described collagen types I and III and transforming growth factor ß. The characterization of the general protein composition of the extracellular matrix in various regions of clubfoot may help in understanding the pathogenesis of this anomaly and, thus, contribute to the development of more efficacious therapeutic approaches.

Proteomic analysis of post-nuclear supernatant fraction and percoll-purified membranes prepared from brain cortex of rats exposed to increasing doses of morphine.

BACKGROUND: Proteomic analysis was performed in post-nuclear supernatant (PNS) and Percoll-purified membranes (PM) prepared from fore brain cortex of rats exposed to increasing doses of morphine (10-50 mg/kg) for 10 days. RESULTS: In PNS, the 10 up (↑)- or down (↓)-regulated proteins exhibiting the largest morphine-induced change were selected, excised manually from the gel and identified by MALDI-TOF MS/MS: 1-(gi|148747414, Guanine deaminase), ↑2.5×; 2-(gi|17105370, Vacuolar-type proton ATP subunit B, brain isoform), ↑2.6×; 3-(gi|1352384, Protein disulfide-isomerase A3), ↑3.4×; 4-(gi|40254595, Dihydropyrimidinase-related protein 2), ↑3.6×; 5-(gi|149054470, N-ethylmaleimide sensitive fusion protein, isoform CRAa), ↑2.0×; 6-(gi|42476181, Malate dehydrogenase, mitochondrial precursor), ↑1.4×; 7-(gi|62653546, Glyceraldehyde-3-phosphate dehydrogenase), ↑1.6×; 8-(gi|202837, Aldolase A), ↑1.3×; 9-(gi|31542401, Creatine kinase B-type), ↓0.86×; 10-(gi|40538860, Aconitate hydratase, mitochondrial precursor), ↑1.3×. The identified proteins were of cytoplasmic (1, 4, 5, 7, 9), cell membrane (2), endoplasmic reticulum (3) and mitochondrial (6, 8, 10) origin and 9 of them were significantly increased, 1.3-3.6×. The 4 out of 9 up-regulated proteins (4, 6, 7, 10) were described as functionally related to oxidative stress; the 2 proteins participate in genesis of apoptotic cell death.In PM, the 18 up (↑)- or down (↓)-regulated proteins were identified by LC-MS/MS and were of plasma membrane [Brain acid soluble protein, ↓2.1×; trimeric Gß subunit, ↓2.0x], myelin membrane [MBP, ↓2.5×], cytoplasmic [Internexin, ↑5.2×; DPYL2, ↑4.9×; Ubiquitin hydrolase, ↓2.0×; 60S ribosomal protein, ↑2.7×; KCRB, ↓2.6×; Sirtuin-2, ↑2.5×; Peroxiredoxin-2, ↑2.2×; Septin-11, ↑2.2×; TERA, ↑2.1×; SYUA, ↑2.0×; Coronin-1A, ↓5.4×] and mitochondrial [Glutamate dehydrogenase 1, ↑2.7×; SCOT1, ↑2.2×; Prohibitin, ↑2.2×; Aspartate aminotransferase, ↓2.2×] origin. Surprisingly, the immunoblot analysis of the same PM resolved by 2D-ELFO indicated that the "active", morphine-induced pool of Gß subunits represented just a minor fraction of the total signal of Gß which was decreased 1.2x only. The dominant signal of Gß was unchanged. CONCLUSION: Brain cortex of rats exposed to increasing doses of morphine is far from being adapted. Significant up-regulation of proteins functionally related to oxidative stress and apoptosis suggests a major change of energy metabolism resulting in the state of severe brain cell "discomfort" or even death.

Insulin signaling and mitochondrial phenotype of skeletal muscle are programmed in utero by maternal diabetes.

Maternal diabetes may influence glucose metabolism in adult offspring, an area with limited research on underlying mechanisms. Our study explored the impact of maternal hyperglycemia during pregnancy on insulin resistance development. Adult female Sprague-Dawley rats from control and diabetic mothers were mated, and their female offspring were monitored for 150 days. The rats were euthanized for blood and muscle samples. Maternal diabetes led to heightened insulin levels, increased HOMA-IR, elevated triglycerides, and a raised TyG index in adult offspring. Muscle samples showed a decreased protein expression of AMPK, PI3K, MAPK, DRP1, and MFF. These changes induced intergenerational metabolic programming in female pups, resulting in insulin resistance, dyslipidemia, and glucose intolerance by day 150. Findings highlight the offspring's adaptation to maternal hyperglycemia, involving insulin resistance, metabolic alterations, the downregulation of insulin signaling sensors, and disturbed mitochondrial morphology. Maintaining maternal glycemic control emerges as crucial in mitigating diabetes-associated disorders in adult offspring.

Comprehensive proteomic analysis of human dentin.

Proteomic analysis of the human body is a significant recent scientific endeavour. In this study, we investigated the proteomic profile of human dentin using modern analytical and mass spectrometric techniques. Five healthy permanent human molars from five adults were cut, pulverized, denaturated with guanidine buffer, and demineralized with EDTA buffer. The extracted proteins were analysed by gel electrophoresis (SDS-PAGE and two-dimensional gel electrophoresis), digested with trypsin, and separated by liquid chromatography/high-resolution tandem mass spectrometry. We identified 289 proteins with high confidence, 90 of which had not been previously detected in human dentin. Nine (currently hypothetical) proteins were identified for the first time in an actual human sample. The proteins have a variety of functions, including calcium-ion binding, formation of the extracellular matrix, formation of the cytoskeleton, cytoskeletal protein binding, immune response, and transport. In conclusion, this is the first use of two-dimensional electrophoresis for investigating human dentin.

Study of saiga horn using high-performance liquid chromatography with mass spectrometry.

The saiga horns have been investigated the using of modern analytic methods. High-performance liquid chromatography (HPLC) with mass-spectrometric (MS and MS/MS) detection and polyacrylamide gel electrophoresis (PAGE) were used. It could be concluded that basic proteins of the saiga horns are keratins and collagen. The basic representation protein in all samples is keratin type I microfibrillar (from sheep), keratin type II microfibrillar (from sheep), collagen type I (α(1)) (from bovine) and collagen type I (α(2)) (from bovine). Free amino acids we determined in all samples are nontreated by enzyme.

The possible role of hypoxia in the affected tissue of relapsed clubfoot.

Our aim was to study the expression of hypoxia-related proteins as a possible regulatory pathway in the contracted side tissue of relapsed clubfoot. We compared the expression of hypoxia-related proteins in the tissue of the contracted (medial) side of relapsed clubfoot, and in the tissue of the non-contracted (lateral) side of relapsed clubfoot. Tissue samples from ten patients were analyzed by immunohistochemistry and image analysis, Real-time PCR and Mass Spectrometry to evaluate the differences in protein composition and gene expression. We found a significant increase in the levels of smooth muscle actin, transforming growth factor-beta, hypoxia-inducible factor 1 alpha, lysyl oxidase, lysyl oxidase-like 2, tenascin C, matrix metalloproteinase-2, matrix metalloproteinase-9, fibronectin, collagen types III and VI, hemoglobin subunit alpha and hemoglobin subunit beta, and an overexpression of ACTA2, FN1, TGFB1, HIF1A and MMP2 genes in the contracted medial side tissue of clubfoot. In the affected tissue, we have identified an increase in the level of hypoxia-related proteins, together with an overexpression of corresponding genes. Our results suggest that the hypoxia-associated pathway is potentially a factor contributing to the etiology of clubfoot relapses, as it stimulates both angioproliferation and fibroproliferation, which are considered to be key factors in the progression and development of relapses.

A Method for Analysis of Nitrotyrosine-Containing Proteins by Immunoblotting Coupled with Mass Spectrometry.

Nitrotyrosine formation is caused by presence of reactive oxygen and nitrogen species. Nitration is a very selective process leading to specific modification of only a few tyrosines in protein molecule. 2D electrophoresis and western blotting techniques coupled with mass spectrometry are common methods used in analysis of proteome. Here we describe protocol for analysis of peroxynitrite-induced protein nitration in isolated mitochondria. Mitochondrial proteins are separated by 2D electrophoresis and transferred to nitrocellulose membrane. Membranes are then incubated with antibodies against nitrotyrosine. Positive spots are compared with corresponding Coomassie-stained gels, and protein nitration is confirmed with mass spectrometry techniques.

Alterations in the Proteome and Phosphoproteome Profiles of Rat Hippocampus after Six Months of Morphine Withdrawal: Comparison with the Forebrain Cortex.

The knowledge about proteome changes proceeding during protracted opioid withdrawal is lacking. Therefore, the aim of this work was to analyze the spectrum of altered proteins in the rat hippocampus in comparison with the forebrain cortex after 6-month morphine withdrawal. We utilized 2D electrophoretic workflow (Pro-Q® Diamond staining and Colloidal Coomassie Blue staining) which was preceded by label-free quantification (MaxLFQ). The phosphoproteomic analysis revealed six significantly altered hippocampal (Calm1, Ywhaz, Tuba1b, Stip1, Pgk1, and Aldoa) and three cortical proteins (Tubb2a, Tuba1a, and Actb). The impact of 6-month morphine withdrawal on the changes in the proteomic profiles was higher in the hippocampus-14 proteins, only three proteins were detected in the forebrain cortex. Gene Ontology (GO) enrichment analysis of differentially expressed hippocampal proteins revealed the most enriched terms related to metabolic changes, cytoskeleton organization and response to oxidative stress. There is increasing evidence that energy metabolism plays an important role in opioid addiction. However, the way how morphine treatment and withdrawal alter energy metabolism is not fully understood. Our results indicate that the rat hippocampus is more susceptible to changes in proteome and phosphoproteome profiles induced by 6-month morphine withdrawal than is the forebrain cortex.

Impact of three-month morphine withdrawal on rat brain cortex, hippocampus, striatum and cerebellum: proteomic and phosphoproteomic studies.

Opioid addiction is characterized by compulsive drug seeking and taking behavior, which is thought to result from persistent neuroadaptations. However, there is a lack of information about the changes at both the cellular and molecular levels occurring after cessation of drug administration. The aim of our study was to determine alterations of both phosphoproteome and proteome in selected brain regions of the rats (brain cortex, hippocampus, striatum, and cerebellum) 3 months after cessation of 10-day morphine treatment. Phosphoproteome profiling was performed by Pro-Q® Diamond staining. The gel-based proteomic approach accompanied by label-free quantification (MaxLFQ) was used for characterization of proteome changes. The phosphoproteomic analysis revealed the largest change in the hippocampus (14); only few altered proteins were detected in the forebrain cortex (5), striatum (4), and cerebellum (3). The change of total protein composition, determined by 2D electrophoresis followed by LFQ analysis, identified 22 proteins with significantly altered expression levels in the forebrain cortex, 19 proteins in the hippocampus, 12 in the striatum and 10 in the cerebellum. The majority of altered proteins were functionally related to energy metabolism and cytoskeleton reorganization. As the most important change we regard down-regulation of 14-3-3 proteins in rat cortex and hippocampus. Our findings indicate that i) different parts of the brain respond in a distinct manner to the protracted morphine withdrawal, ii) characterize changes of protein composition in these brain parts, and iii) enlarge the scope of evidence for adaptability and distinct neuroplasticity proceeding in the brain of drug-addicted organism.

Accelerated in vitro recellularization of decellularized porcine pericardium for cardiovascular grafts.

An ideal decellularized allogenic or xenogeneic cardiovascular graft should be capable of preventing thrombus formation after implantation. The antithrombogenicity of the graft is ensured by a confluent endothelial cell layer formed on its surface. Later repopulation and remodeling of the scaffold by the patient's cells should result in the formation of living autologous tissue. In the work presented here, decellularized porcine pericardium scaffolds were modified by growing a fibrin mesh on the surface and inside the scaffolds, and by attaching heparin and human vascular endothelial growth factor (VEGF) to this mesh. Then the scaffolds were seeded with human adipose tissue-derived stem cells (ASCs). While the ASCs grew only on the surface of the decellularized pericardium, the fibrin-modified scaffolds were entirely repopulated in 28 d, and the scaffolds modified with fibrin, heparin and VEGF were already repopulated within 6 d. Label free mass spectrometry revealed fibronectin, collagens, and other extracellular matrix proteins produced by ASCs during recellularization. Thin layers of human umbilical endothelial cells were formed within 4 d after the cells were seeded on the surfaces of the scaffold, which had previously been seeded with ASCs. The results indicate that an artificial tissue prepared by in vitro recellularization and remodeling of decellularized non-autologous pericardium with autologous ASCs seems to be a promising candidate for cardiovascular grafts capable of accelerating in situ endothelialization. ASCs resemble the valve interstitial cells present in heart valves. An advantage of this approach is that ASCs can easily be collected from the patient by liposuction.

Growth of vascular smooth muscle cells on collagen I exposed to RBL-2H3 mastocytoma cells.

Remodeling of the peripheral pulmonary vasculature during chronic hypoxia is characterized by accelerated collagenolysis and thickening of the vascular wall. Low molecular weight peptides, products of cleavage by interstitial collagenase and muscular layer in the peripheral pulmonary vessels, are typically present. The aim of this "in vitro" study was to verify that mast cells (RBL-2H3) as a potent source of a variety of biomolecules which can affect vessel wall remodeling are capable of splitting collagen and then facilitating the growth of vascular smooth muscle cells (VSMC). Collagen I was exposed to RBL-2H3 cells cultured 48 hours under normoxic or hypoxic (3% O(2)) conditions and then seeded with VSMC. The VSMC proliferated with the shortest doubling time and reached the highest cell population density on the collagen pre-modified with hypoxic RBL-2H3 cells. This increased growth activity of VSMC was probably due to the fragmentation of collagen by proteases released from RBL-2H3 cells. Absolute amount of collagen fragments was similar in samples exposed to normoxic and hypoxic RBL-2H3 cells, but the concentration of at least one collagen fragment was significantly higher under hypoxic conditions. Mast cells exposed to hypoxia are more capable to split collagen and facilitate the growth of VSMC.

Determination of insoluble avian eggshell matrix proteins.

The organic components of bones and other mineralized tissues have a high impact on the organization and deposition of calcium, and consequently influence the mechanical properties of those tissues. The extractable proteins of avian eggshells have been studied extensively and many of them have been identified; insoluble (non-extractable) proteins have been sparsely studied, however. In the work discussed in this paper we studied EDTA-insoluble proteins by gradual decalcification of eggshell with EDTA. The insoluble proteinaceous films were chemically treated with cyanogen bromide and the mixtures of large fragments obtained were gradually precipitated with salt. The separated fractions were digested with trypsin and analyzed by HPLC-MS-MS (ion trap mass spectrometer). Analysis of the entire eggshell matrix (without precipitation steps) only enabled 6 proteins to be determined (ovocalyxins 32 and 36, ovocleidin 17 and 116, clusterin, and ovalbumin). Pretreatment of the individual eggshell layers and gradual precipitation with salt markedly increased the number of proteins identified - 28 proteins were determined. We identified for the first time collagens I (two chains) and III in the eggshell matrix, and Kunitz-like protease inhibitor as a major shell matrix protein. Besides the above mentioned proteins we can also mention EDIL3, fibronectin, sulfhydryl oxidase, tubulin alpha 1, lysozyme, Dickkopf-related protein 3, keratins, and ovotransferrin. The relative abundances of proteins in all eggshell layers were determined using the exponentially modified protein abundance index (emPAI). In the cuticle layer seven proteins were identified, whereas 16 proteins were described in the palisade layer and 23 in the mammillary layer.