Native Australian plant extracts differentially induce Collagen I and Collagen III in vitro and could be important targets for the development of new wound healing therapies

Diabetic nephropathy (DN) is the most frequent cause of end-stage renal disease. Tubulointerstitial accumulation of lysine 63 (K63)-ubiquitinated (Ub) proteins is involved in the progression of DN fibrosis and correlates with urinary miR-27b-3p downregulation. We explored the renoprotective effect of an inhibitor of K63-Ub (NSC697923), alone or in combination with the ACE-inhibitor ramipril, in vitro and in vivo. Proximal tubular epithelial cells and diabetic DBA/2J mice were treated with NSC697923 and/or ramipril. K63-Ub protein accumulation along with α-SMA, collagen I and III, FSP-1, vimentin, p16INK4A expression, SA-α Gal staining, Sirius Red, and PAS staining were measured. Finally, we measured the urinary albumin to creatinine ratio (uACR), and urinary miR-27b-3p expression in mice. NSC697923, both alone and in association with ramipril, in vitro and in vivo inhibited hyperglycemia-induced epithelial to mesenchymal transition by significantly reducing K63-Ub proteins, α-SMA, collagen I, vimentin, FSP-1 expression, and collagen III along with tubulointerstitial and glomerular fibrosis. Treated mice also showed recovery of urinary miR-27b-3p and restored expression of p16INK4A. Moreover, NSC697923 in combination with ramipril demonstrated a trend in the reduction of uACR. In conclusion, we suggest that selective inhibition of K63-Ub, when combined with the conventional treatment with ACE inhibitors, might represent a novel treatment strategy to prevent the progression of fibrosis and proteinuria in diabetic nephropathy and we propose miR-27b-3p as a biomarker of treatment efficacy.

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Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring

Journal of Cell Science ◽

10.1242/jcs.108.3.985 ◽

1995 ◽

Vol 108 (3) ◽

pp. 985-1002 ◽

Cited By ~ 9

Author(s):

M. Shah ◽

D.M. Foreman ◽

M.W. Ferguson

Keyword(s):

Wound Healing ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Collagen I ◽

Tgf Beta ◽

Early Stages ◽

Collagen Iii ◽

Exogenous Addition ◽

Growth Factor Beta ◽

Beta 2

Exogenous addition of neutralising antibody to transforming growth factor-beta 1,2 to cutaneous wounds in adult rodents reduces scarring. Three isoforms of transforming growth factor-beta (1, 2 and 3) have been identified in mammals. We investigated the isoform/isoforms of TGF-beta responsible for cutaneous scarring by: (i) reducing specific endogenous TGF-beta isoforms by exogenous injection of isoform specific neutralising antibodies; and (ii) increasing the level of specific TGF-beta isoforms by exogenous infiltration into the wound margins. Exogenous addition of neutralising antibody to TGF-beta 1 plus neutralising antibody to TGF-beta 2 reduced the monocyte and macrophage profile, neovascularisation, fibronectin, collagen III and collagen I deposition in the early stages of wound healing compared to control wounds. Treatment with neutralising antibodies to TGF-betas 1 and 2 markedly improved the architecture of the neodermis to resemble that of normal dermis and reduced scarring while the control wounds healed with scar formation. Exogenous addition of neutralising antibody to TGF-beta 1 alone also reduced the monocyte and macrophage profile, fibronectin, collagen III and collagen I deposition compared to control wounds. However, treatment with neutralising antibody to TGF-beta 1 alone only marginally reduced scarring. By contrast, wounds treated with neutralising antibody to TGF-beta 2 alone did not differ from control wounds. Interestingly, exogenous addition of the TGF-beta 3 peptide also reduced the monocyte and macrophage profile, fibronectin, collagen I and collagen III deposition in the early stages of wound healing and markedly improved the architecture of the neodermis and reduced scarring. By contrast, wounds treated with either TGF-beta 1 or with TGF-beta 2 had more extracellular matrix deposition in the early stages of wound healing but did not differ from control wounds in the final quality of scarring. This study clearly demonstrates isoform specific differences in the role of TGF-betas in wound healing and cutaneous scarring. TGF-beta 1 and TGF-beta 2 are implicated in cutaneous scarring. This study also suggests a novel therapeutic use of exogenous recombinant, TGF-beta 3 as an anti-scarring agent.

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Physiological regulation of extracellular matrix collagen and elastin in the arterial wall of rats by noradrenergic tone and angiotensin II

Journal of the Renin-Angiotensin-Aldosterone System ◽

10.1177/1470320311414752 ◽

2011 ◽

Vol 13 (1) ◽

pp. 19-28 ◽

Cited By ~ 5

Author(s):

Houcine Dab ◽

Kamel Kacem ◽

Rafik Hachani ◽

Nadra Dhaouadi ◽

Wassim Hodroj ◽

...

Keyword(s):

Extracellular Matrix ◽

Angiotensin Ii ◽

Vascular Diseases ◽

Vascular Wall ◽

Collagen I ◽

Ang Ii ◽

Physiological Regulation ◽

Collagen Iii ◽

Wistar Kyoto

The interactions between the effects of the sympathetic nervous system (SNS) and angiotensin II (ANG II) on vascular extracellular matrix (ECM) synthesis were determined in rats. The mRNA and protein content of collagen I, collagen III and elastin in the abdominal aorta (AA) and femoral artery (FA) was investigated in Wistar–Kyoto rats treated for 5 weeks with guanethidine, a sympathoplegic, losartan, an ANG II AT1 receptor (AT1R) blocker, or both. The effects of noradrenaline (NE) and ANG II on collagen III and elastin mRNA, and the receptor involved, were tested in cultured vascular smooth muscle cells (VSMCs) in vitro. Guanethidine increased collagen types I and III and decreased elastin, while losartan had an opposite effect, although without effect on collagen III. The combination of treatments abrogated changes induced by simple treatment with collagen I and elastin, but increased collagen III mRNA in AA and not in FA. NE stimulated collagen III mRNA via β receptors and elastin via α1 and α2 receptors. ANG II stimulated collagen III but inhibited elastin mRNA via AT1R. Overall, SNS and ANG II exert opposite and antagonistic effects on major components of ECM in the vascular wall. This may be of relevance for the choice of a therapeutic strategy in vascular diseases.

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Aspirin Reduces Cardiac Interstitial Fibrosis by Inhibiting Erk1/2-Serpine2 and P-Akt Signalling Pathways

Cellular Physiology and Biochemistry ◽

10.1159/000487972 ◽

2018 ◽

Vol 45 (5) ◽

pp. 1955-1965 ◽

Cited By ~ 3

Author(s):

Xuelian Li ◽

GuoYuan Wang ◽

MuGe QiLi ◽

HaiHai Liang ◽

TianShi Li ◽

...

Keyword(s):

Cardiac Fibrosis ◽

Interstitial Fibrosis ◽

Smooth Muscle Actin ◽

Pressure Overload ◽

Collagen I ◽

Signalling Pathways ◽

Collagen Iii ◽

The Impact

Background/Aims: Cardiac interstitial fibrosis is an abnormality of various cardiovascular diseases, including myocardial infarction, hypertrophy, and atrial fibrillation, and it can ultimately lead to heart failure. However, there is a lack of practical therapeutic approaches to treat fibrosis and reverse the damage to the heart. The purpose of this study was to investigate the effect of long-term aspirin administration on pressure overload–induced cardiac fibrosis in mice and reveal the underlying mechanisms of aspirin treatment. Methods: C57BL/6 mice were subjected to transverse aortic constriction (TAC), and treated with 10 mg·kg-1·day-1 of aspirin for 4 weeks. Masson staining and a collagen content assay were used to detect the effects of aspirin on cardiac fibrosis in vivo and in vitro. Western blot and qRT-PCR were applied to examine the impact of aspirin on extracellular signal-regulated kinases (Erks), p-Akt/β-catenin, SerpinE2, collagen I, and collagen III levels in the mice heart. Results: Aspirin significantly suppressed the expression of α-smooth muscle actin (α-SMA; 1.19±0.19-fold) and collagen I (0.95±0.09-fold) in TAC mice. Aspirin, at doses of 100 and 1000 µM, also significantly suppressed angiotensin II-induced α-SMA and collagen I in cultured CFs. The enhanced phosphorylation of Erk1/2 caused by TAC (p-Erk1, 1.49±0.19-fold; p-Erk2, 1.96±0.68-fold) was suppressed by aspirin (p-Erk1, 1.04±0.15-fold; p-Erk2, 0.87±0.06-fold). SerpinE2 levels were suppressed via the Erk1/2 signalling pathway following treatment with aspirin (1.36±0.12-fold for TAC; 1.06±0.07-fold for aspirin+TAC). The p-Akt and β-catenin levels were also significantly inhibited in vivo and in vitro. Conclusions: Our study reveals a novel mechanism by which aspirin alleviates pressure overload-induced cardiac interstitial fibrosis in TAC mice by suppressing the p-Erk1/2 and p-Akt/β-catenin signalling pathways.

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In Vitro Gingival Wound Healing Activity of Extracts from Reynoutria japonica Houtt Rhizomes

Pharmaceutics ◽

10.3390/pharmaceutics13111764 ◽

2021 ◽

Vol 13 (11) ◽

pp. 1764

Author(s):

Izabela Nawrot-Hadzik ◽

Adam Matkowski ◽

Artur Pitułaj ◽

Barbara Sterczała ◽

Cyprian Olchowy ◽

...

Keyword(s):

Wound Healing ◽

Laser Scanning ◽

Immunocytochemical Staining ◽

Confocal Laser ◽

Phytochemical Analysis ◽

Chinese Medicinal Herb ◽

Traditional Use ◽

Reynoutria Japonica ◽

Collagen Iii

Rhizomes of Reynoutria japonica Houtt. are a traditional Chinese medicinal herb (Polygoni cuspidati rhizoma, hu zhang) used for treatment of numerous diseases including wound healing support. The aim of this study was to provide evidence for the value of this herbal drug’s traditional use as a gingival healing treatment as well as to obtain the most active extract. In vitro studies were performed using primary human gingival fibroblasts (HGFs) with determination of viability (MTT assay), cell proliferation (the confocal laser scanning microscope (CLSM) was used to visualize histone 3 expression), cell migration (wound healing assay), and evaluation of the expression of collagen type III (immunocytochemical staining) after incubation with extracts from R. japonica rhizomes (25% or 40% ethanol or 60% acetone). In addition to these extracts, commercial dental rinse (containing chlorhexidine digluconate 0.2%) was tested as the gold standard of choice for gum healing in dental practice. The studied extracts were qualitatively and quantitatively characterized using the validated HPLC/DAD/ESI-HR-QTOF-MS method. Total phenols and tannins content were determined using the Folin–Ciocalteu assay. Low concentration of all extracts after 24 h incubation caused significant increase in HGF viability. This effect was most pronounced at a concentration of 50 µg/mL, which was selected for further experiments. All extracts (at 50 µg/mL) stimulated HGF to proliferate, migrate, and increase collagen III synthesis, but with different strength. The highest stimulated proliferation and migration activity was observed after incubation with 25% EtOH, which according to phytochemical analysis may be related to the highest content of resveratrol and an appropriate composition of procyanidins. The 25% EtOH extract from R. japonica rhizomes appears to be a promising gingival wound healing agent worthy of animal and clinical trials.

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Integrin alpha 2 beta 1 is upregulated in fibroblasts and highly aggressive melanoma cells in three-dimensional collagen lattices and mediates the reorganization of collagen I fibrils.

The Journal of Cell Biology ◽

10.1083/jcb.115.5.1427 ◽

1991 ◽

Vol 115 (5) ◽

pp. 1427-1436 ◽

Cited By ~ 246

Author(s):

C E Klein ◽

D Dressel ◽

T Steinmayer ◽

C Mauch ◽

B Eckes ◽

...

Keyword(s):

Wound Healing ◽

Connective Tissue ◽

Cell Lines ◽

Three Dimensional ◽

Melanoma Cells ◽

Human Melanoma ◽

Collagen I ◽

Collagen Gels ◽

Melanoma Cell Lines

The ability of cultured human fibroblasts to reorganize and contract three dimensional collagen I gels is regarded as an in vitro model for the reorganization of connective tissue during wound healing. We investigated whether adhesion receptors of the integrin family are involved. It was found that synthesis and transcription of the alpha 2 beta 1 integrin (but not of alpha 1 beta 1 or alpha 3 beta 1) is selectively upregulated when fibroblasts are seeded into type I collagen gels. Time course experiments revealed that high synthetic levels of alpha 2 beta 1 parallel the gel contraction process and return to "baseline" levels after the contraction has subsided. Furthermore, function-blocking mAbs directed to the alpha 2 and beta 1 chain of integrins inhibited gel contraction. Remodelling of connective tissue can be important for tumor cells during invasion and formation of metastases. Therefore, we tested human melanoma cell lines for this function. Five out of nine melanoma lines contracted collagen gels in vitro. Among these, two highly aggressive melanoma cell lines (MV3 and BLM) most efficiently contracted gels almost reaching the rate of normal adult fibroblasts. In these cells, synthesis of alpha 2 beta 1 was also significantly upregulated when seeded into collagen I gels. Moreover, function blocking anti-alpha 2 in conjunction with anti-beta 1 chain mAbs completely inhibited gel contraction for several days. Other melanoma cells (530) with lower metastatic potential which were not able to contract gels, showed no induction of alpha 2 beta 1 synthesis in gel culture. Our results suggest an important role of integrin alpha 2 beta 1 in the contraction of collagen I by normal diploid fibroblasts during wound healing and in the reorganization of collagen matrices by highly aggressive human melanoma cells.

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Impact of the Transfersome Delivered Human Growth Hormone on the Dermal Fibroblast Cells

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666190809120333 ◽

2019 ◽

Vol 20 (14) ◽

pp. 1194-1202 ◽

Cited By ~ 3

Author(s):

Minoo Azimi ◽

Mahvash Khodabandeh ◽

Abdolkhalegh Deezagi ◽

Fatemeh Rahimi

Keyword(s):

Growth Hormone ◽

Cell Migration ◽

Dermal Fibroblast ◽

Skin Permeation ◽

Human Growth ◽

Fibroblast Cell ◽

Skin Aging ◽

Collagen I ◽

Collagen Iii

Objective: Transfersomes are highly flexible vesicles that are capable of passing through pores smaller than their own sizes due to their elastochemical characteristics, and thus play a key role in drug delivery to the skin. Methods: In this study, we used transdermal delivery of growth hormone-encapsulated transferosomes (F1 and F2) as antiaging strategy, with the resulting effects being subsequently evaluated. The size, distribution and zeta potential of the particles, together with the in vitro skin permeation and biological activity of the growth hormone loaded onto the transfersomes were measured. Results: The data demonstrated that treatment of fibroblasts with encapsulated hGH increased cell migration, proliferation and collagen I and III gene expression. According to our results, the maximum amount of growth hormone that passes through the skin during a 24 h time period was 489.54 and 248.46 ng/cm3, for the F1 and F2 transfersomes, respectively. In addition, it was determined that F1 formula as the more efficient carrier, showed no toxicity against cells. With regard to fibroblasts, as one of the most important cells involved in collagen synthesis, skin aging and wound healing, concentrations of growth hormone encapsulated in transferosomes that had an effect on fibroblast growth and division, were determined. The results demonstrated that effective concentrations of the encapsulated growth hormone increased the expression of collagen I and collagen III genes. Conclusion: Furthermore, analyzing the rate of fibroblast cell migration showed that migration increased significantly at 700 ng/ml growth hormone concentrations, as compared to that of the control.

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Atropa Belladonna L. Water Extract: Modulator of Extracellular Matrix Formation in Vitro and in Vivo

Physiological Research ◽

10.33549/physiolres.932223 ◽

2012 ◽

pp. 241-250 ◽

Cited By ~ 7

Author(s):

P. GÁL ◽

T. VASILENKO ◽

I. KOVÁČ ◽

M. KOSTELNÍKOVÁ ◽

J. JAKUBČO ◽

...

Keyword(s):

Wound Healing ◽

Skin Wound ◽

Dermal Fibroblasts ◽

Histological Evaluation ◽

Control Group ◽

Atropa Belladonna ◽

Skin Wound Healing ◽

Collagen Iii

Previously, we found that treatment of cutaneous wounds with Atropa belladonna L. (AB) revealed shortened process of acute inflammation as well as increased tensile strength and collagen deposition in healing skin wounds (Gál et al. 2009). To better understand AB effect on skin wound healing male Sprague-Dawley rats were submitted to one round full thickness skin wound on the back. In two experimental groups two different concentrations of AB extract were daily applied whereas the control group remained untreated. For histological evaluation samples were removed on day 21 after surgery and stained for wide spectrum cytokeratin, collagen III, fibronectin, galectin-1, and vimentin. In addition, in the in vitro study different concentration of AB extract were used to evaluate differences in HaCaT keratinocytes proliferation and differentiation by detection of Ki67 and keratin-19 expressions. Furthermore, to assess ECM formation of human dermal fibroblasts on the in vitro level fibronectin and galectin-1 were visualized. Our study showed that AB induces fibronectin and galectin-1 rich ECM formation in vitro and in vivo. In addition, the proliferation of keratinocytes was also increased. In conclusion, AB is an effective modulator of skin wound healing. Nevertheless, further research is needed to find optimal therapeutic concentration and exact underlying mechanism of action.

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Impairment of the Hif-1α regulatory pathway in Foxn1-deficient (Foxn1−/−) mice affects the skin wound healing process

10.1101/2020.08.04.237388 ◽

2020 ◽

Author(s):

Sylwia Machcinska ◽

Marta Kopcewicz ◽

Joanna Bukowska ◽

Katarzyna Walendzik ◽

Barbara Gawronska-Kozak

Keyword(s):

Wound Healing ◽

Transforming Growth Factor ◽

Healing Process ◽

Transforming Growth Factor Β ◽

Skin Wound ◽

Wound Healing Process ◽

Limiting Factor ◽

Skin Wound Healing ◽

Collagen Iii

ABSTRACTHypoxia and hypoxia-regulated factors [e. g., hypoxia-inducible factor-1α (Hif-1α), factor inhibiting Hif-1α (Fih-1), thioredoxin-1 (Trx-1), aryl hydrocarbon receptor nuclear translocator 2 (Arnt-2)] have essential roles in skin wound healing. Using Foxn1−/− mice that can heal skin injuries in a unique scarless manner, we investigated the interaction between Foxn1 and hypoxia-regulated factors. The Foxn1−/− mice displayed impairments in the regulation of Hif-1α, Trx-1 and Fih-1 but not Arnt-2 during the healing process. An analysis of wounded skin showed that the skin of the Foxn1−/− mice healed in a scarless manner, displaying rapid re-epithelialization and an increase in transforming growth factor β (Tgfβ-3) and collagen III expression. An in vitro analysis revealed that Foxn1 overexpression in keratinocytes isolated from the skin of the Foxn1−/− mice led to reduced Hif-1α expression in normoxic but not hypoxic cultures and inhibited Fih-1 expression exclusively under hypoxic conditions. These data indicate that in the skin, Foxn1 affects hypoxia-regulated factors that control the wound healing process and suggest that under normoxic conditions, Foxn1 is a limiting factor for Hif-1α.

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Collagenase H is Crucial for Isolation of Rat Pancreatic Islets

Cell Transplantation ◽

10.3727/096368913x668654 ◽

2014 ◽

Vol 23 (10) ◽

pp. 1187-1198 ◽

Cited By ~ 15

Author(s):

Atsushi Fujio ◽

Kazutaka Murayama ◽

Youhei Yamagata ◽

Kimiko Watanabe ◽

Takehiro Imura ◽

...

Keyword(s):

Mass Spectrometry ◽

Immunohistochemical Analysis ◽

Collagen I ◽

Islet Isolation ◽

Pancreatic Islet Isolation ◽

Collagen Iii ◽

Target Matrix ◽

And Function

The role(s) of collagenase G (ColG) and collagenase H (ColH) during pancreatic islet isolation remains controversial, possibly due to the enzyme blends used in the previous studies. We herein examined the role of ColG and ColH using highly pure enzyme blends of recombinant collagenase of each subtype. Rat pancreases were digested using thermolysin, together with ColG, ColH, or ColG/ColH ( n = 9, respectively). No tryptic-like activity was detected in any components of the enzyme blends. The efficiency of the collagenase subtypes was evaluated by islet yield and function. Immunohistochemical analysis, in vitro collagen digestion assay, and mass spectrometry were also performed to examine the target matrix components of the crucial collagenase subtype. The islet yield was highest in the ColG/ColH group (4,101 ± 460 islet equivalents). A substantial number of functional islets (2,811 ± 581 islet equivalents) was obtained in the ColH group, whereas no islets were retrieved in the ColG group. Mass spectrometry demonstrated that ColH reacts with collagen I and III. In the immunohistochemical analysis, both collagen I and III were located in exocrine tissues, although collagen III expression was more pronounced. The collagen digestion assay showed that collagen III was more effectively digested by ColH than by ColG. The present study reveals that ColH is crucial, while ColG plays only a supporting role, in rat islet isolation. In addition, collagen III appears to be one of the key targets of ColH.

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