Total Hydroxyproline Content Research Articles

A Novel Food Bore Protein Hydrogel with Silver Ions for Promoting Burn Wound Healing.

Hydrogels have emerged as a promising option for treating local scald wounds due to their unique physical and chemical properties. This study aims to evaluate the efficacy of ovalbumin/gelatin composite hydrogels in repairing deep II-degree scald wounds using a mouse dorsal skin model. Trauma tissues collected at various time points are analyzed for total protein content, hydroxyproline content, histological features, and expression of relevant markers. The results reveal that the hydrogel accelerates the healing process of scalded wounds, which is 17.27% higher than the control group. The hydrogel treatment also effectively prevents wound enlargement and redness of the edges caused by infection during the initial stage of scalding. The total protein and hydroxyproline content of the treated wounds are significantly elevated. Additionally, the hydrogel up-regulates the expression of VEGF (a crucial angiogenic factor) and down-regulates CD68 (a macrophage marker). In summary, this study provides valuable insights into the potential of multifunctional protein-based hydrogels in wound healing.

Topical Application of Siberian Pine Essential Oil Formulations Enhance Diabetic Wound Healing.

This study aimed to develop novel topical formulations based on a natural component (0.5% of Siberian pine essential oil) and to assess its wound-healing capacity through macroscopic, histopathological, and biochemical examination. The phytochemical profile of Pinus sibirica essential oil (PSEO) and rheological analysis and safety potential of formulations were determined. The wound-healing effect was evaluated on an excision wound model in diabetic Wistar albino rats randomly divided into the following groups topically treated with (1) untreated, (2) 1% silver sulfadiazine, (3) ointment base, (4) gel base, (5) PSEO ointment, and (6) PSEO gel. Formulations containing PSEO were stable and safe for skin application. Three weeks of treatment with both PSEO formulations (ointment and gel) led to a significant reduction in wound size (98.14% and 96.28%, respectively) and a remarkably higher level of total hydroxyproline content (9.69 µg/mg and 7.26 µg/mg dry tissue, respectively) relative to the control group (65.97%; 1.81 µg/mg dry tissue). These findings were in correlation with histopathological results. Topically applied PSEO formulations were associated with a significant reduction in most of the measured pro-oxidants and enhanced activity of the antioxidant defense system enzymes (p < 0.05). Our findings showed that gel and ointment with PSEO demonstrated significant wound-repairing capabilities in the excision wound model.

Changes in the indicators of connective tissue metabolism in the blood serum of experimental rats under the conditions of modeling the development of degenerative processes in paravertebral muscles

Low back pain is a common health problem. To deepen the understanding of the pathogenesis of the disease, experimental studies on animals with modeling of the pathological process are necessary. Objective. Based on the analysis of biochemical markers of connective tissue metabolism in the blood serum of laboratory rats, the applicability of the studied models of degenerative muscle tissue damage to study the relationship between this condition and the development of disorders in spinal motor segments was assessed. Methods. Two models of reproduction of degenerative processes in the paravertebral muscles of white rats were tested: I (n = 5) — alimentary (diet-induced) obesity, by keeping it for 3 months on a high-calorie diet; II (n = 5) — ischemia, by tying the large rectus muscles of the back with suture material (45 days). Control group (n = 5) — intact animals of similar age and sex. The content of glycoproteins, total chondroitin sulfates (CS), hexosamines, protein-bound hexoses, seroglycoides, fractional distribution and total content of hydroxyproline and glycosaminoglycan sulfates (GAGs) were investigated in the blood serum of rats. The results. In the blood serum of rats of groups I and II, a significant increase compared to the control level of glycoproteins was determined, with a greater effect in the ischemia model, but no significant changes of protein-bound hexoses, hexosamines and CS were recorded. The level of inflammatory markers (sialic acids and seroglycoides) in the blood serum of animals of both groups did not differ significantly from the control, and changes in the parameters of hydroxyproline (except for the slightly changed protein-bound fraction) and GAGs were significant only for the ischemia model. Conclusions. Based on the analysis of biochemical markers of connective tissue metabolism in rats of groups I and II, changes characteristic of degenerative processes were determined, with a greater manifestation in the ischemia model. No significant increase in biochemical markers of inflammation was recorded. Both models can be used to reproduce dystrophic processes in osteochondrosis.

The vulnerability of the human taenia coli to alterations in total collagen within the colon of the elderly

The structure of the colonic wall relies on collagen, distributed within the submucosa and the muscularis externa. A recent analysis of total collagen in human ascending colon (AC) suggests that the muscularis externa is more susceptible to age-related increases in collagen among the elderly. However, it is not clear if this change also occurs in the descending colon (DC) or if the circular and longitudinal muscle layers are similarly affected in either region of colon. The aim of this study is to determine the total collagen content in the DC and its distribution between the circular muscle (CM) and taenia coli (TC) of the AC and DC of adults and compare the same with tissue from the elderly. Masson’s trichrome and Picrosirius red were used to assess total collagen content in the AC and DC; aged 22 – 91 years. Macroscopically normal AC from 22 patients (adults: 22–60 years; 6 male, 6 female; elderly: 70 – 91 years; 6 male, 4 female) and DC from 23 patients (adults: 23–63 years; 6 male, 7 female; elderly: 66 – 88 years; 6 male, 4 female) were obtained following surgery for non-obstructed bowel cancer. The total hydroxyproline content in DC samples was also evaluated. In the DC, tinctorial staining demonstrated an increased occurrence of total collagen fibres in the submucosa of the elderly (159.8 ± 9.6 in elderly vs. 126.9 ± 6.1 in the adults; p 0.05) and in the muscularis externa (respectively 37.4 ± 4.1 vs. 18.8 ± 2.4; p 0.01). In the adult AC and DC, there were no statistically significant differences in the amount of collagen within the CM and TC. In the elderly, the total collagen fibres within the TC was greater in the AC (mean grey intensity: 63.4 ± 3.9% in the elderly vs. 36.6 ± 1.6% in adults; p 0.05) and DC (mean grey intensity: respectively, 59.82 ± 2.4 vs. 40.2 ± 0.9%; p 0.05). In both AC and DC of the elderly samples, several thickened collagen fibrils were microscopically identified within the TC infiltrating to the myenteric plexus. In the TC of the elderly AC, the total collagen fibres were increased by approximately 4% compared to that of the DC. The total collagen concentration in the elderly DC assessed by hydroxyproline assay was increased by approximately 15% compared to the adult. Sex related differences were not found when data combined. We concluded that the total collagen content in the muscularis externa particularly of the TC of human colon increases with age. The subtle change in collagen distribution with age between AC and DC may differentially affect the tensile strength of the colon.

Characterization, antimicrobial and cytotoxic activity of polymer blends based on chitosan and fish collagen

This study aims to produce, characterize, and assess the antimicrobial activity and cytotoxicity of polymer blends based on chitosan (CT) and fish collagen (COL) produced by different precipitation methods. Polymer blends were obtained in alkaline (NaOH), saline (NaCl), and alkaline/saline (NaOH/NaCl) solutions with different CT:COL concentration ratios (20:80, 50:50, and 80:20). The polymer blends were characterized by various physicochemical methods and subsequently evaluated in terms of their in vitro antimicrobial and cytotoxicity activity. In this study, the degree of chitosan deacetylation was 82%. The total hydroxyproline and collagen content in the fish matrix was 47.56 mg. g−1 and 394.75 mg. g−1, respectively. The highest yield was 44% and was obtained for a CT:COL (80:20) blend prepared by precipitation in NaOH. High concentrations of hydroxyproline and collagen in the blends were observed when NaOH precipitation was used. Microbiological analysis revealed that the strains used in this work were sensitive to the biomaterial; this sensitivity was dose-dependent and increased with increasing chitosan concentration in the products. The biocompatibility test showed that the blends did not reduce the viability of fibroblast cells after 48 h of culture. An analysis of the microbiological activity of the all-polymer blends showed a decrease in the values of minimal inhibitory concentration (MIC) and minimal bactericidal concentrations (MBC) for S. aureus and P. aeruginosa. The blends showed biocompatibility with NIH-3T3 murine fibroblast cells and demonstrated their potential for use in biomedical applications such as wound healing, implants, and scaffolds.

Modulation of H4K16Ac levels reduces pro-fibrotic gene expression and mitigates lung fibrosis in aged mice.

Histone H4 lysine16 acetylation (H4K16Ac) modulates chromatin structure by serving as a switch from a repressive to a transcriptionally active state. This euchromatin mark is associated with active transcription. In this study, we investigated the effects of H4K16Ac on the expression of pro-fibrotic genes in lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) and in an aging murine model of lung fibrosis.Methods: The lung tissues and fibroblasts from human IPF/non-IPF donors and from aged mice with/without bleomycin induced lung fibrosis were used in this study. The H4K16Ac levels were examined by immunohistochemistry or western blots. RNA silencing of H4K16Ac acetyltransferase Mof was used to reduce H4K16Ac levels in IPF fibroblasts. The effects of reduced H4K16Ac on pro-fibrotic gene expression were examined by western blots and real-time PCR. The association of H4K16Ac with these genes' promoter region were evaluated by ChIP assays. The gene expression profile in siRNA Mof transfected IPF cells were determined by RNA-Seq. The impact of H4K16Ac levels on lung fibrosis was evaluated in an aging murine model.Results: Aged mice with bleomycin induced lung fibrosis showed increased H4K16Ac levels. Human lung fibroblasts with siRNA Mof silencing demonstrated reduced H4K16Ac, and significantly down-regulated profibrotic genes, such as α-smooth muscle actin (α-SMA), collagen I, Nox4, and survivin. ChIP assays confirmed the associations of these pro-fibrotic genes' promoter region with H4K16Ac, while in siRNA Mof transfected cells the promoter/H4K16Ac associations were depleted. RNA-seq data demonstrated that Mof knockdown altered gene expression and cellular pathways, including cell damage and repair. In the aging mice model of persistent lung fibrosis, 18-month old mice given intra-nasal siRNA Mof from week 3 to 6 following bleomycin injury showed improved lung architecture, decreased total hydroxyproline content and lower levels of H4K16Ac.Conclusions: These results indicate a critical epigenetic regulatory role for histone H4K16Ac in the pathogenesis of pulmonary fibrosis, which will aid in the development of novel therapeutic strategies for age-related diseases such as IPF.

Conditioned medium from a human adipose-derived stem cell line ameliorates inflammation and fibrosis in a lung experimental model of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis is a chronic, progressive parenchymal lung disease that results in fibrogenesis and the conditioned medium from adipose-derived mesenchymal stem cells (CM-ADSCs) has been shown to be efficacious in pulmonary fibrosis animal models. The aim of the present study is to evaluate the effect of CM-ADSCs on lung inflammation and fibrosis in a Bleomycin (BLM)-induced pulmonary fibrosis model. CM-ADSCs safety and toxicity were evaluated in Sprague Dawley rats and no adverse effects were observed. Six-week-old female C57BL/6J mice were employed in the BLM-induced pulmonary fibrosis model and were divided into four groups: Group 1 (Sham): animals were kept without BLM and treatment, Group 2 (Control): BLM with vehicle DMEM, Group 3: 10 μg/kg CM-ADSCs and Group 4: 100 μg/kg CM-ADSCs. Body weight, fibrosis and inflammation histological analyses, mRNA and protein pro-inflammatory cytokine, and total hydroxyproline content calculation were performed in all groups upon sacrifice. The 100 μg/kg CM-ADSCs showed a significant increase in mean body weight compared to Controls. CM-ADSCs doses resulted in the amelioration of fibrosis, as seen by Masson's Trichrome-staining, Ashcroft scoring, and Sirius red-staining. Compared to Controls, inflammation was also significantly reduced in CM-ADSCs-treated mice, with reduced F4/80 macrophage antigen staining, TNF-α mRNA and IL-6 and IL-10 protein levels. Total hydroxyproline content was found significantly reduced in both groups of CM-ADSCs-treated mice. Overall, our study shows that the CM-ADSCs is safe and efficient against pulmonary fibrosis, as it significantly reduced inflammation and fibrosis, with the larger dose of 100 μg/kg CM-ADSCs being the most efficient one.

Formulation and Evaluation of Helichrysum italicum Essential Oil-Based Topical Formulations for Wound Healing in Diabetic Rats.

As proper wound management is crucial to reducing morbidity and improving quality of life, this study evaluated for the first time the wound healing potential of H. italicum essential oil (HIEO) prepared in the form of ointment and gel in streptozotocin-induced diabetic wound models in rats. After creating full-thickness cutaneous wounds, forty-eight diabetic rats were divided into six groups: (1) negative control; (2) positive control; (3) ointment base; (4) gel base; (5) 0.5% HIEO ointment (6) 0.5% HIEO gel. Wound healing potential was determined by the percentage of wound contraction, hydroxyproline content, redox status, and histological observation. A significant decrease in the wound size was observed in animals treated with HIEO formulations compared with other groups. The HIEO groups also showed a higher level of total hydroxyproline content, and more pronounced restitution of adnexal structures with only the underlying muscle defect indicating the incision site. Hence, our results legitimate the traditional data of the pro-healing effect of HIEO because HIEO in both formulations such as gel and ointment exhibited the significant wound repairing effect in the incision wound model.

Evaluation of in vivo Wound Healing Activity of Ursolic Acid Rich Chloroform Extract of Hedyotis herbacea Linn Ointment

Aim: This study evaluates the effect of in vivo wound healing potential of Ursolic acid rich chloroform extract of Hedyotis herbacea Linn ointment using incision and excision wound model.&#x0D; Study Design: Wound healing potentials of Hedyotis herbacea were analysed by Incision and excision wound model.&#x0D; Place of Study: Nandha College of Pharmacy, Erode, Tamilnadu.&#x0D; Methodology: Hedyotis herbacea was subjected to extraction (cold maceration), with solvents of increasing polarity. All the extracts were estimated for the presence of phytoconstituents by HPTLC. As the study has been focused on the phytoconstituent based biological activity, the Ursolic acid rich chloroform extract was chosen for the study of wound healing activity. The Chloroform extract of Hedyotis herbacea was incorporated into ointment base, to prepare the ointment. The ointments prepared with Chloroform extract (2.5% and 5%) of Hedyotis herbacea (CEHH) were subjected for evaluation of excision and incision wound model. Wistar albino rats were divided into four groups each consisting of six animals; group I (left untreated) considered as untreated control, group II treated with 5% w/w povidone-iodine ointment served as standard, group III treated with CEHH 2.5% w/w ointment, and group IV treated with CEHH 5% w/w ointment were considered as test groups. All the treatments were given once daily. Wound healing effect was assessed by the rate of wound contraction, level of total protein, Hydroxy proline, Hexosamine and Hexuronic acid and histopathology studies in an excision wound model. Tensile strength was also measured in both excision and incision wound.&#x0D; Results: Wound healing activity of CEHH 5% w/w ointment treated group was greater than CEHH 2.5% w/w and untreated groups in both excision and incision wound model. The high rate of wound contraction (*P&lt; 0.001), high tensile strength (*P&lt; 0.001), and elevated total protein, Hydroxyproline, Hexosamine, and Hexuronic acid content were observed in animals treated with CEHH ointments when compared to the untreated control group of animals. Histopathological studies of the CEHH ointments treated groups also revealed the effectiveness in wound healing.&#x0D; Conclusion: These results justified the claimed traditional use of the Hedyotis herbacea as wound healing plant.

Vitrified Human Umbilical Arteries as Potential Grafts for Vascular Tissue Engineering.

The development of a biological based small diameter vascular graft (d < 6mm), that can be properly stored over a long time period at - 196°C, in order to directly be used to the patients, still remains a challenge. In this study the decellularized umbilical arteries (UAs) where vitrified, evaluated their composition and implanted to a porcine model, thus serving as vascular graft. Human UAs were decellularized using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) detergents. Then, vitrified with vitrification solution 55 (VS55) solution, remained for 6months in liquid nitrogen and their extracellular matrix composition was compared to conventionally cryopreserved UAs. Additionally, total hydroxyproline, sulphated glycosaminoglycan and DNA content were quantified in all samples. Finally, the vitrified umbilical arteries implanted as common carotid artery interposition graft to a porcine animal model. Decellularized and vitrified UAs characterized by proper preservation of extracellular matrix proteins and tissue architecture, whereas conventionally cryopreserved samples exhibited a disorganized structure. Total hydroxyproline content was preserved, although sulphated glycosaminoglycan and DNA contents presented significantly alterations in all samples. Implanted UAs successfully recellularized and remodeled as indicated by the histological analysis. Decellularized and vitrified UAs retained their structure function properties and can be possible used as an alternative source for readily accessible small diameter vascular grafts.

Сравнительный анализ биохимических показателей при ревматических заболеваниях взрослых и детей в норме и патологии

Collagen is an intercellular substance of connective tissue, it plays an important role in the physiological processes of the body. Collagen consists of three polypeptide chains twisted in a spiral and in order to stabilize this structure, hydroxyproline is needed, its hydroxyl groups are involved in the formation of hydrogen bonds between the chains of polypeptides. The peculiarity of collagen is that 14% of the amino acids it contains are hydroxyproline, an amino acid that is not contained in other proteins. In this regard, the content of hydroxyproline in blood serum was used to assess the metabolism of collagen in the body. We determined the content of hydroxyproline fractions in blood serum. Calcium, fibrinogen, erythrocyte settling rate and alkaline phosphatase level in children and adults with joint disease were also determined. The control group included healthy children and adults. The total hydroxyproline content in children with the disease was 9-11% lower than in healthy children. In adults with joint diseases, the total hydroxyproline content was 11% higher than in healthy patients. In children and adults with rheumatic diseases, the peptide-bound hydroxyproline content increased, while the protein-bound hydroxyproline content decreased in comparison with the control group control parameters. No significant changes in the content of calcium and fibrinogen were noted, but the level of alkaline phosphatase and the rate of erythrocyte sedimentation was increased, which is typical for people with inflammatory diseases.

Effect of Ficus carica L. leaves extract loaded gold nanoparticles against cisplatin-induced acute kidney injury

BackgroundCisplatin (CisPt) is one of the most widely used and highly effective drugs for the treatment of various solid tumors, unfortunately acute kidney injury (AKI) is considered one of its side effects through several mechanisms including production of reactive oxygen species (ROS), pro-inflammatory and pro-fibrotic cytokines. Due to the poor effect of AKI therapy, the use of nanoparticles loaded with natural extracts for delivering to the kidney molecules are desirable. AimThis study aims to investigate the effectiveness of different concentrations of gold nanoparticles (Au-NPs) as a carrier for Ficus carica L. (Fig) leaves extract against CisPt induced AKI. MethodsSeventy male albino rats were used and divided into seven groups. After the experimental period, blood was withdrawn, serum was separated for determination of urea, creatinine, homocystein (Hcy) and folic acid while reduced glutathione (GSH), nitric oxide (NO), malondialdehyde (MDA), total antioxidant capacity (TAC) and hydroxyproline content (Hyp) were evaluated in kidney tissue homogenate. ResultsCisPt induced AKI in rats and results in a significant increase in the levels of serum urea, creatinine, Hcy and kidney Hyp, lipid peroxidation along with a significant reduction of kidney GSH, NO and TAC compared to the control rats. Treatment with Au-NPs and Fig extract particularly in a ratio of (3:2) respectively was shown to improve renal functions with efficient capacity in scavenging ROS and reduced AKI severity. ConclusionAu-NPs enhanced the anti-oxidative properties of the Fig extract in targeting kidney damaged tissue and reduced oxidative toxicity induced by CisPt.

MON-476 Growth Hormone and the Glomerular Podocyte: Studies of Podocyte-Specific Growth Hormone Receptor Gene Deletion in Mice

Previous research has suggested a role for growth hormone (GH) in the development of kidney disease. Evidence of increased glomerular size and sclerosis in bovine GH (bGH) overproducing animals suggests that excess GH action may cause or exacerbate glomerular damage. Within the glomerulus, the podocyte plays the role of forming a filtration barrier called the slit diaphragm. This cell is known to be dysfunctional in many models of nephropathy and is also a direct target of GH. To explore the mechanisms by which GH impacts the glomerulus, we have developed a transgenic mouse, the podocyte-specific GH receptor gene-disrupted mouse (podGHR-/-). These mice were generated on a C57BL/6J background using Cre-Lox transgenic methods. To examine the effects of high GH levels on the podocyte, these mice were crossed with bovine GH (bGH) overproducing mice and studied at several time points over the course of a year, alongside wild-type control mice and podGHR-/- mice lacking bGH overexpression. Various metabolic and physiologic assays were performed on these mice, including glomerular filtration rate (GFR) testing, glucose and insulin tolerance testing, body composition analysis, and urine & serum analysis. Histological analyses were also performed. Podocyte-specific GHR gene-deletion was associated with increased GFR in male podGHR-/- mice at 54 weeks of age, and with decreased GFR in male podGHR-/- mice with bGH overexpression at 54 weeks of age. PodGHR-/- male mice exhibited significantly decreased total kidney hydroxyproline content at 54 weeks of age as well as decreased glomerular trichrome stain deposition. Both male and female podGHR-/- mice showed decreased glomerular periodic acid Schiff stain deposition at 54 weeks of age. These results suggest a role for podocyte GH activity in the regulation of GFR in the aging and diseased kidney, as well as a role for GH in directing the deposition of collagen and glycosaminoglycans in glomerulus via the podocyte.

Assessment of physico-chemical traits related to eating quality of young dairy bull beef at different ageing times using Raman spectroscopy and chemometrics

Raman spectroscopy and chemometrics were investigated for the prediction of eating quality related physico-chemical traits of Holstein-Friesian bull beef. Raman spectra were collected on the 3rd, 7th and 14th days post-mortem. A frequency range of 1300–2800cm−1 was used for partial least squares (PLS) modelling. PLS regression (PLSR) models for the prediction of WBSF and cook loss achieved an R2CV of 0.75 with RMSECV of 6.82 N and an R2CV of 0.77 with RMSECV of 0.97%w/w respectively. For the prediction of intramuscular fat, moisture and crude protein content, R2CV values were 0.85, 0.91 and 0.70 with RMSECV of 0.52%w/w, 0.39%w/w and 0.38%w/w respectively. An R2CV of 0.79 was achieved for the prediction of both total collagen and hydroxyproline content, while for collagen solubility the R2CV was 0.88. All samples (100%) from 15- and 19-month old bulls were correctly classified using PLS discriminant analysis (PLS-DA), while 86.7% of samples from different muscles (longissimus thoracis, semitendinosus and gluteus medius) were correctly classified. In general, PLSR models using Raman spectra on the 3rd day post-mortem had better prediction performance than those on the 7th and 14th days. Raman spectroscopy and chemometrics have potential to assess several beef physical and chemical quality traits.

Adipose tissue-derived stem cells ameliorates dermal fibrosis in a mouse model of scleroderma.

To investigate the therapeutic potential of adipose-derived stem cells (ADSCs) for limited cutaneous scleroderma (LS) in mouse models. ADSCs were isolated from pathogen-free female C57BL/6 mice and LS was induced in wild type (WT) C57BL/6 mice via daily injection of bleomycin (0.1mL×300μg/mL) for 4 weeks; then the ADSCs were subcutaneously injected into the dorsal area in the model treatment group, and 100μL of phosphate-buffered saline (PBS) solution was injected into the same site in the model control group. Green fluorescent protein (GFP) was used to track the cells using an invivo imaging system on days 7, 14, 21, and 28 after transplantation. All mice were sacrificed and histologic analyses were performed after 4 weeks, and the skin thickness, collagen deposition and the total content of hydroxyproline were evaluated. Additionally, immunohistochemistry were performed to compare the tissue expression and distribution of TGF-β1 and VEGF between the ADSCs treatment group and the treatment control group. WT C57BL/6 LS mouse model were successfully established and GFP invivo fluorescence imaging showed that the translated ADSCs survived at the local for at least 4 weeks. Compared with the control group, the ADSCs treatment group significantly attenuated bleomycin-induced dermal fibrosis, reduced the skin thickness and the total content of hydroxyproline (P<0.05). The ADSCs treatment group displayed significantly lower levels of TGF-β1 and higher levels of VEGF than the control group (P<0.05). ADSCs may provide a feasible and practical treatment for autoimmune diseases such as LS and ameliorate dermal fibrosis.

Paquinimod reduces skin fibrosis in tight skin 1 mice, an experimental model of systemic sclerosis

BackgroundSystemic Sclerosis (SSc) is an autoimmune disease characterized by vascular and immune dysfunction. A hallmark of SSc is the excessive accumulation of extracellular matrix in the skin and in internal organs. There is a high and unmet medical need for novel therapies in this disease. The pathogenesis of SSc is complex and still poorly understood, but the innate immune system has emerged as an important factor in the disease. SSc patients show increased numbers of macrophages/monocytes in the blood and in the skin compared to healthy individuals and these cells are important sources of profibrotic cytokines and chemokines. Paquinimod belongs to a class of orally active quinoline-3-carboxamide (quinoline) derivatives with immunomodulatory properties and has shown effects in several models of autoimmune/inflammatory disorders. Paquinimod is currently in clinical development for treatment of SSc. The immunomodulatory effects of paquinimod is by targeting the myeloid cell compartment via the S100A9 protein. ObjectiveIn this study we investigate whether targeting of myeloid cells by paquinimod can effect disease development in an experimental model of SSc, the tight skin 1 (Tsk-1) mouse model. MethodsSeven weeks old female B6.Cg-Fbn1Tsk/J (Tsk-1) mice were treated with vehicle or paquinimod at the dose of 5 or 25mg/kg/day in the drinking water for 8 weeks. The effect of paquinimod on the level of skin fibrosis and on different subpopulations within the myeloid cell compartment in skin biopsies were evaluated by using histology, immunohistochemisty, a hydroxyproline assay and real-time PCR. Furthermore, the level of IgG in serum from treated animals was also analysed. The statistical analyses were performed using Mann-Whitney nonparametric two tailed rank test. ResultsThe results show that treatment with paquinimod reduces skin fibrosis measured as reduction of skin thickness and decreased number of myofibroblasts and total hydroxyproline content. The effect on fibrosis was associated with a polarization of macrophages in the skin from a pro-fibrotic M2 to a M1 phenotype. Paquinimod treatment also resulted in a reduced TGFβ-response in the skin and an abrogation of the increased auto-antibody production in this SSc model. ConclusionsPaquinimod reduces skin fibrosis in an experimental model of SSc, and this effect correlates with local and systemic effects on the immune system.

Rapamycin Attenuates Age-associated Changes in Tibialis Anterior Tendon Viscoelastic Properties.

Rapamycin extends mouse life span, but the extent to which rapamycin prevents aging-associated changes in specific tissues remains unclear. Stiffness increases and collagen turnover decreases in mouse tendon with aging; thus, our aim was to determine the effect of long-term rapamycin treatment on the mechanical and structural properties of tendons from old mice. Tendons were harvested from female UM-HET3 mice maintained on a standard chow diet for 4 (adult) or 22 (old) months or fed chow containing polymer-encapsulated rapamycin (eRAPA) from 9 to 22 months of age (old RAPA). Stiffness was twofold higher for tendons of old compared with adult mice, but in old RAPA mice, tendon stiffness was maintained at a value not different from that of adults. Additionally, expression of collagen decreased, expression of matrix metalloproteinase-8 increased, and total hydroxyproline content trended toward decreased levels in tendons of old eRAPA-fed mice compared with controls. Finally, age-associated calcification of Achilles tendons and accompanying elevations in expression of chondrocyte and osteoblast markers were all lower in old eRAPA-fed mice. These results suggest that long-term administration of rapamycin alters the molecular pathways responsible for aging of tendon extracellular matrix, resulting in tissue that is structurally and mechanically similar to tendons in adult mice.

Preclinical Efficacy of TGF-Beta Receptor I Kinase Inhibitor, Galunisertib, in Myelofibrosis

Background: Bone marrow fibrosis is associated with poor prognosis in myeloproliferative neoplasms (MPN). Both polycythemia vera (PV) and essential thrombocythemia (ET) patients are at high risk for transformation into myelofibrosis. Although JAK2 inhibitors are effective treatment for MPNs, they are unable to alleviate the fibrotic phenotype. Since TGFβ has been implicated in the development of fibrosis in this disease, we determined the expression of TGF-β pathway in human MPN samples and also evaluated the anti-fibrotic effect of a clinically relevant TGF-β receptor inhibitor (Galunisertib, LY2157299) in MPN mouse models.Results: To determine the pathobiology of TGF-β signaling in MPNs, we analyzed gene expression profiles of 93 MPN patient derived neutrophils and 11 controls. We determined that the TGF-β1 isoform was the dominant ligand expressed in these samples and was significantly increased in primary myelofibrosis(PMF) samples (p=0.001). Examination of downstream SMAD pathways revealed that SMAD2 was the dominant expressed effector in MPN samples and was also significantly increased in PMF samples (p=0.005).Galunisertib(LY2157299) is a specific inhibitor of TGF-β receptor I kinase and abrogates the phosphorylation of SMAD2. Next, we determined preclinical efficacy of LY2157299 in a mouse model generated by transgenic expression of JAK2V617F using the vav-1 promoter. These mice display a PV-like phenotype with elevated blood cell counts at the age of 6 weeks and develop bone marrow and spleen fibrosis at 25-30 weeks. Microarray analysis of gene expression in JAK2V617F and control total bone marrow revealed significant increase in TGF-β1 expression in JAK2V617F animals and immunohistochemistry demonstrated increased SMAD2 activation consistent with primary human data. Thirty-week old mice with established myelofibrosis were treated with LY2157299 daily for 4 weeks. A significant decrease in fibrosis was assessed quantitatively by total hydroxyproline content in bone marrow after treatment (mean hydroxyproline content 5ug/ml in control mice vs 2.1ug/ml in treated mice, p=0.005). To validate the efficacy of inhibition of this pathway, we next treated 50 week-old JAK2V617F transgenic mice with a murine antibody against TGFβ1 (IMC-TR1, LY3022859) at 40mg/kg 3 times per week for 4 weeks. This treatment also resulted in significant decrease in fibrosis (p=0.02).For further verification, we used the MPLW515L transplantation mouse model that develops rapid thrombocytosis, leukocytosis, splenomegaly and myelofibrosis. For in vivo studies, LY2157299 was orally administered daily from day 12 after transplantation for 14 consecutive days. Through in vitro studies of MSCs in transplant recipients with WT-MPL, we found that LY2157299 effectively antagonizes the stimulatory effect of TGFβ on collagen I (p<0.05) and collagen III (p<0.01) production in a dose-dependent manner. Treatment of MPLW515L mice with LY2157299 for 14 days significantly decreased reticulin fiber formation compared with those treated with vehicle control (p<0.05). MSCs derived from LY2157299-treated mice also showed significantly less collagen production compared to MSCs from vehicle-treated MPLW515L mice (p<0.05). Although no reduction in spleen size was observed in MPLW515L-transplanted mice, a significant reduction in the peripheral WBC count was observed after treatment with LY2157299 at 150mg/kg (p<0.05).Conclusions: These results indicate that TGFβ-receptor blockade with Galunisertib inhibits TGFβ-induced collagen production and attenuates the fibrotic phenotype in MPN mouse models. DisclosuresLevine:CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.

Biochemical parameters of serum and lumbar intervertebral discs in the experimental rabbits after denucleation

Development of medical science toward understanding of the processes taking places in cases of degenerative diseases of the musculoskeletal system is impossible without modeling of pathological conditions in laboratory animals. Objective: To investigate the changes in biochemical parameters of metabolism in tissues of intervertebral discs and serum in rabbits with modeled degenerative and destructive process in lumbar intervertebral discs. Methods: The model included mechanical denucleation of one nucleus pulposus of intervertebral disc and damage to the cranial apophysis of the vertebral body with the growth zone in the lumbar spine of 3-month-old rabbits (weight of 1 300–1 500 g). 60 days after the operation we determined the content of total hydroxyproline, hexosamine, total glycosaminoglycans (GAG), sialic acid and tyrosine in tissues of intervertebral discs. Before and 60 days after modeling we evaluated the content of glycoproteins, total protein, fractional composition of glycosaminoglycans sulfates (GAGs) and hydroxyproline in blood serum. Results: There was reliable activation of cartilage collagen metabolism of intervertebral discs with the increase of hydroxyproline content and inhibition of proteoglycan metabolism with a decrease of GAG content and hexosamine concentration of sialic acids and non-collagenous proteins in intervertebral discs of experimental animals compared to control group. Degenerative processes in experimental rabbits prevailed over reparative ones. In blood serum of experimental animals there was revealed reliable increase in the total content of GAGs to 25.76 % and hydroxyproline to 16.87 %, as well as decrease to 42.61 % of activity of alkaline phosphatase and increase of 282.95 % of activity of acid phosphatase. Conclusion: The model created leads to the development of degenerative-dystrophic process on the 60th day after the operation.

The use of ultrasound-stimulated contrast agents as an adjuvant for collagenase therapy in chronic total occlusions

To investigate the effectiveness of combining collagenase and ultrasound-stimulated microbubble (USMB) treatments in reducing the mechanical force required for crossing a guidewire through CTOs. Experiments were conducted on ex vivo specimens of a rabbit femoral artery CTO model (n=45 total samples). Four primary groups were employed: control (n=6), collagenase only (n=15), USMB only (1 MHz frequency) (n=5), and collagenase+USMB (n=19). In one set of experiments the force required to puncture through CTO samples was measured and it was found that the puncture force was 2.31-fold lower for the combined treatment group relative to the comparable collagenase-only group (p<0.05). In a second set of experiments, the total protein and hydroxyproline content of the supernatant solution adjacent to the CTO was analysed. Significantly higher hydroxyproline levels were measured in collagenase+USMB treated CTOs (0.065 g/mL) compared to collagenase (0.030 g/mL), USMB (0.003 g/mL) and control (0.004 g/mL) (p<0.05), indicating that the combined treatment augmented collagenase degradation. Ultrasound-stimulated microbubbles improved the effectiveness of collagenase in reducing the force required to cross experimental CTOs. This new approach may have the potential to reduce treatment times and improve the success rates of emerging collagenase-based treatments of CTO.