scholarly journals Systemic Sclerosis Serum Significantly Impairs the Multi-Step Lymphangiogenic Process: In Vitro Evidence

2019 ◽  
Vol 20 (24) ◽  
pp. 6189 ◽  
Author(s):  
Mirko Manetti ◽  
Eloisa Romano ◽  
Irene Rosa ◽  
Bianca Saveria Fioretto ◽  
Serena Guiducci ◽  
...  
Keyword(s):  
Wound Healing ◽  
Systemic Sclerosis ◽  
Vascular System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Boyden Chamber ◽  
Vegf Receptor ◽  
Lymphatic Capillary ◽  
Matrigel Invasion ◽  
Gene And Protein Expression

In systemic sclerosis (SSc), the possible involvement of lymphatic microcirculation and lymphangiogenesis has traditionally been overshadowed by the greater emphasis placed on dysfunctional blood vascular system and angiogenesis. In the present in vitro study, we explore for the first time whether the SSc microenvironment may interfere with lymphangiogenesis, a complex, multi-step process in which lymphatic microvascular endothelial cells (LMVECs) sprout, migrate, and proliferate to generate new lymphatic capillaries. Normal human adult dermal LMVECs from three donors were treated with serum from SSc patients (n = 8), serum from healthy individuals (n = 8), or recombinant human vascular endothelial growth factor (VEGF)-C as a positive control for lymphangiogenesis. Cell proliferation, Boyden chamber Matrigel chemoinvasion, wound healing capacity, and lymphatic capillary morphogenesis on Geltrex were assayed. VEGF-C serum levels were measured by enzyme-linked immunosorbent assay. Gene and protein expression levels of the lymphangiogenic orchestrators VEGF receptor-3 (VEGFR-3)/Flt-4 and neuropilin-2 (NRP-2) were determined by real-time PCR and Western blotting, respectively. Conditioning with SSc serum significantly inhibited LMVEC proliferation, Matrigel invasion, and wound healing capacity with respect to healthy serum. The ability of LMVECs to form lymphatic tubes on Geltrex was also severely compromised in the presence of SSc serum. VEGF-C levels were comparable in SSc and healthy sera. Treatment with SSc serum resulted in a significant downregulation of both VEGFR-3/Flt-4 and NRP-2 mRNA and protein levels. In SSc, the pathologic environment severely hampers every lymphangiogenesis step, likely through the reduction of pro-lymphangiogenic VEGFR-3/NRP-2 co-receptor signaling. The impairment of the lymphangiogenic process opens a new scenario underlying SSc vascular pathophysiology, which is worth investigating further.

scholarly journals TH17 cells expressing CD146 are significantly increased in patients with Systemic sclerosis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Amira Gabsi ◽  
Xavier Heim ◽  
Akram Dlala ◽  
Asma Gati ◽  
Haifa Sakhri ◽  
...  
Keyword(s):  
Systemic Sclerosis ◽  
Th17 Cells ◽  
Vascular System ◽  
Adaptive Immune Response ◽  
Autoimmune Disorder ◽  
Soluble Form ◽  
Adaptive Immune ◽  
Th17 Lymphocytes

AbstractSystemic sclerosis (SSc) is an autoimmune disorder characterized by vascular damage, excessive fibrosis and abnormal T cells immune-regulation. CD146 is an adhesion molecule essentially expressed in the vascular system, but also on TH17 lymphocytes. In view of the recently described role of CD146 in SSc, we hypothesized an involvement of CD146 positive TH17 cells in this disease. Compared to healthy controls, we showed that both soluble form of CD146 (sCD146), and IL17A levels were increased in patients with SSc with a positive correlation between both factors. A significant increase in TH17 cells attested by an increase of RORγT, IL17A mRNA and CD4+ IL17A+ cell was observed in patients with SSc. Interestingly, the percentage of TH17 cells expressing CD146 was higher in patients with SSc and inversely correlated with pulmonary fibrosis. In vitro experiments showed an augmentation of the percentage of TH17 cells expressing CD146 after cell treatment with sCD146, suggesting that, in patients the increase of this sub-population could be the consequence of the sCD146 increase in serum. In conclusion, TH17 cells expressing CD146 could represent a new component of the adaptive immune response, opening the way for the generation of new tools for the management of SSc.

VEGF-VEGF Receptor 1 (Flt-1) Interactions Significantly Modulate Endothelial Progenitor Cell (EPC) Migration but Have Only Minor Effects on the Migration of Differentiated Endothelial Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3229-3229
Author(s):  
Bin Li ◽  
Amylynn A. Teleron ◽  
Charles Lin ◽  
Pampee P. Young
Keyword(s):  
Wound Healing ◽  
Endothelial Cells ◽  
Blood Vessel ◽  
Vegf Receptor ◽  
Blocking Antibody ◽  
Blood Vessel Formation ◽  
Migration Assay ◽  
Vessel Formation ◽  
Cellular Expression

Abstract There is a growing body of data demonstrating that vasculogenesis, whereby bone marrow -derived circulating progenitor cells (EPCs) home to sites of neovascularization, results in significant contribution to blood vessel formation during tumor growth, ischemic injury and wound healing. Vascular Endothelial Growth Factor (VEGF) has recently been shown to augment vasculogenesis. In the current study, we examined if VEGF/VEGF Receptor (VEGFR) interactions are important for EPC recruitment. Both VEGFR1 (flt-1) and VEGFR2 (flk-1) are strongly expressed, as detected by immunofluorescent and FACS analysis, on EPCs obtained by ex vivo expansion of human peripheral blood. In a modified Boyden chamber migration assay, EPCs showed dose dependent migration to VEGF. To examine receptor specificity, EPCs were preincubated with receptor blocking anti-VEGFR1 or -VEGFR2 prior to the migration assay. Level of inhibition by VEGFR1 blocking antibody was commensurate with blocking VEGFR2. Furthermore, migration in response to a VEGFR1-specific agonist, PlGF, was comparable to that induced by VEGF and was completely ablated by preincubation with VEGFR1 blocking antibody. By contrast, differentiated endothelial cells had diminished migration in response to PlGF as compared to VEGF. Furthermore, blocking VEGFR1 only mildly disrupted VEGF-induced migration of differentiated endothelial cells in vitro. Hence, unlike differentiated endothelial cells, EPC migration in vitro was mediated by both VEGF receptors. By quantitative RT-PCR, we examined the level of VEGFR1 and VEGFR2 mRNA transcripts in EPCs versus differentiated endothelial cells. VEGFR1 transcripts in EPCs were expressed 3-fold higher than in differentiated endothelial cells. VEGFR2 and neuropilin transcript levels in EPCs, however, were lower than in differentiated endothelial cells. These results suggest that VEGF/VEGFR1 interactions are important in EPC migration in vitro. We have subcloned VEGFR1 cDNA into a retrovirus vector and have shown by western blot that we can direct increased cellular expression of VEGFR1. In further experiments, we will examine the role of VEGFR1 in human EPC recruitment using murine xenotransplant models of hindlimb ischemia and wound healing. These studies will provide valuable insight towards developing EPCs as cellular therapy to augment blood vessel formation.

scholarly journals A Tobacco-Derived Thymosinβ4 Concatemer Promotes Cell Proliferation and Wound Healing in Mice

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Rylosona Janarthini ◽  
Xiaolei Wang ◽  
Lulu Chen ◽  
Lei Gao ◽  
Lingxia Zhao
Keyword(s):  
Wound Healing ◽  
Quantitative Polymerase Chain Reaction ◽  
Great Promise ◽  
Enzyme Linked Immunosorbent Assay ◽  
Migration Assay ◽  
Direct Administration ◽  
Polymerase Chain ◽  
Endothelial Migration ◽  
Unit Repeat

Thymosinβ4 (Tβ4) is a peptide that is known to play important roles in protection, regeneration, and remodeling of injured tissues in humans, and that shows great promise in a range of clinical applications. However, current strategies to Tβ4 are insufficient to meet growing demand and have a number of limitations. In this current study we investigated whether expression of recombinant Tβ4 in plants, specifically in tobacco (Nicotiana tabacum) leaves, represents an effective approach. To address this question, a 168 bpTβ4gene optimized for tobacco codon usage bias was constitutively expressed in tobacco as a 4-unit repeat concatemer, fused to a polyhistidine tag. Quantitative polymerase chain reaction and Western blot analyses were used to verify4×Tβ4expression in 14 transgenic tobacco lines and enzyme-linked immunosorbent assay analysis indicated 4×Tβ4 protein concentrations as high as 3 μg/g of fresh weight in the leaves. We observed that direct administration of tobacco-derived Tβ4 was more effective than Tβ4 either obtained commercially or derived from expression inEscherichia coliat promoting splenocyte proliferationin vitroand wound healing in mice through an endothelial migration assay. This study provides new insights into the development of plant-derived therapeutic proteins and their application by direct administration.

scholarly journals In Vitro Wound Healing Potential of Photobiomodulation Is Possibly Mediated by Its Stimulatory Effect on AKT Expression in Adipose-Derived Stem Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Naresh K. Rajendran ◽  
Nicolette N. Houreld ◽  
Heidi Abrahamse
Keyword(s):  
Stem Cells ◽  
Wound Healing ◽  
Power Density ◽  
Western Blotting ◽  
Akt Signaling ◽  
Enzyme Linked Immunosorbent Assay ◽  
Adipose Derived Stem Cells ◽  
Cell Models ◽  
Trypan Blue Exclusion

Increasing evidence suggests that adipose-derived stem cells (ADSCs) serve as a therapeutic approach for wound healing. The aim of this study was to determine the effect of photobiomodulation (PBM) on antioxidant enzymes in ADSCs. Four ADSC cell models, namely, normal, wounded, diabetic, and diabetic wounded, were irradiated with 660 nm (fluence of 5 J/cm2 and power density of 11.2 mW/cm2) or 830 nm (fluence of 5 J/cm2 and power density of 10.3 mW/cm2). Nonirradiated cells served as controls. Cell morphology and wound migration were determined using light microscopy. Cell viability was determined by the trypan blue exclusion assay. The enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of antioxidants (superoxide dismutase (SOD), catalase (CAT), and heme oxygenase (HMOX1)). AKT activation and FOXO1 levels were determined by immunofluorescence and western blotting. The gaps (wound) in PBM-treated wounded and diabetic wounded cell models closed faster than the controls. PBM treatment significantly increased antioxidant levels in all cell models. This reflects that oxidative stress is reduced on the counterpart of increased antioxidant levels. This might be due to the activation of the AKT signaling pathway as evidenced by the increased AKT signals via western blotting and immunofluorescence. This data suggests that PBM promotes wound healing by increasing antioxidant levels by activating AKT signaling.

scholarly journals The distribution pattern and growth factor level in platelet-rich fibrin incorporated skin-derived mesenchymal stem cells: An in vitro study

2020 ◽  
Vol 13 (10) ◽  
pp. 2097-2103
Author(s):  
Igo Syaiful Ihsan ◽  
Deya Karsari ◽  
Nora Ertanti ◽  
Aristika Dinaryanti ◽  
Alexander Patera Nugraha ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wound Healing ◽  
Mesenchymal Stem Cells ◽  
Growth Factor ◽  
Distribution Pattern ◽  
Enzyme Linked Immunosorbent Assay ◽  
Platelet Rich Fibrin ◽  
Group A ◽  
Group D

Background and Aim: A skin wound in an animal must be cared for to prevent further health issues. Platelet-rich fibrin (PRF) and skin-derived mesenchymal stem cells (SMSCs) have been reported to have potential in increasing the rate of wound healing. This study aimed to analyze the distribution patterns and levels of platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), and transforming growth factor-β (TGF-β) in PRF incorporated with SMSCs. Materials and Methods: This study employed a true experiment (in vitro) design with post-test only performed in the control group alone. PRF and SMSCs were extracted from the blood and skin of 16 rabbits. SMSCs were characterized using immunocytochemistry to examine clusters of differentiation for 45, 73, 90, and 105. PRF was incorporated into the SMSCs and then divided into four groups (N=32/n=8): Group A (PRF only), Group B (PRF+SMSCs, incubated for 1 day), Group C (PRF+SMSCs, incubated for 3 days), and Group D (PRF+SMSCs, incubated for 5 days). Scanning electron microscopy was used to examine the distribution pattern of SMSCs between groups. The supernatant serum (Group A) and supernatant medium culture (Group D) were collected for the measurement of PDGF, IGF, VEGF, and TGF-β using an enzyme-linked immunosorbent assay sandwich kit. An unpaired t-test was conducted to analyze the differences between Groups A and D (p<0.01). Results: Group D had the most morphologically visible SMSCs attached to the PRF, with elongated and pseudopodia cells. There was a significant difference between the levels of growth factor in Groups A and D (p=0.0001; p<0.01). Conclusion: SMSCs were able to adhere to and distribute evenly on the surface of PRF after 5 days of incubation. The PRF incorporated SMSCs contained high levels of PDGF, IGF, VEGF, and TGF- β, which may prove to have potential in enhancing wound healing.

scholarly journals Thrombin Preconditioning of Extracellular Vesicles Derived from Mesenchymal Stem Cells Accelerates Cutaneous Wound Healing by Boosting Their Biogenesis and Enriching Cargo Content

2019 ◽  
Vol 8 (4) ◽  
pp. 533 ◽  
Author(s):  
Dong Kyung Sung ◽  
Yun Sil Chang ◽  
Se In Sung ◽  
So Yoon Ahn ◽  
Won Soon Park
Keyword(s):  
Wound Healing ◽  
Extracellular Vesicles ◽  
Therapeutic Efficacy ◽  
Tube Formation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Umbilical Cord ◽  
Cutaneous Wound Healing ◽  
Cutaneous Wound

The aim of this study was to determine the optimal preconditioning regimen for the wound healing therapeutic efficacy of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs). To this end, we compared various preconditioning regimens for both the quantitative and qualitative production of MSC-derived EVs, and their therapeutic efficacy for proangiogenic activity in vitro and cutaneous wound healing in vivo. After preconditioning with thrombin (40 U), H2O2 (50 μM), lipopolysaccharide (1 μg/mL), or hypoxia (10% O2), EV secretion was assessed quantitatively by measuring production per cell and protein quantification, and qualitatively by measuring a proteome profiler and an enzyme-linked immunosorbent assay (ELISA) contained within EVs. The therapeutic efficacy of EVs was assessed in vitro by proliferation, migration and tube formation assays of human umbilical cord blood endothelial cells (HUVECs), and in vivo by quantification of cutaneous wound healing. Thrombin preconditioning optimally boosted EV production and enriched various growth factors including vascular endothelial growth factor and angiogenin contained within EVs compared to other preconditioning regimens. Thrombin preconditioning optimally enhanced proliferation, the migration and tube formation of HUVECs in vitro via pERK1/2 and pAKT signaling pathways, and cutaneous wound healing in vivo compared to other preconditioning regimens. Thrombin preconditioning exhibited optimal therapeutic efficacy compared with other preconditioning regimens in promoting proangiogenic activity in vitro and in enhancing cutaneous wound healing in vivo. These preconditioning regimen-dependent variations in therapeutic efficacy might be mediated by boosting EV production and enriching their cargo content.

Evidence for a Derangement of the Microvascular System in Patients with a Very Early Diagnosis of Systemic Sclerosis

2017 ◽  
Vol 44 (8) ◽  
pp. 1190-1197 ◽  
Author(s):  
Inês Chora ◽  
Eloisa Romano ◽  
Mirko Manetti ◽  
Celestina Mazzotta ◽  
Raquel Costa ◽  
...  
Keyword(s):  
Wound Healing ◽  
Systemic Sclerosis ◽  
Early Diagnosis ◽  
Microvascular Dysfunction ◽  
Serum Levels ◽  
Tube Formation ◽  
Serum Samples ◽  
Angiogenic Potential ◽  
Migration Capacity

Objective.To investigate whether patients with a very early diagnosis of systemic sclerosis (VEDOSS) may already present circulating markers and in vitro signs of microvascular dysfunction.Methods.Serum samples were obtained from 55 patients with systemic sclerosis (SSc), 25 patients with VEDOSS, and 55 matched healthy controls (HC). Serum levels of pan-vascular endothelial growth factor (VEGF) and soluble neuropilin-1 (sNRP-1) were measured by ELISA. Human dermal microvascular endothelial cells (H-MVEC) were cultured and stimulated with SSc, VEDOSS, and HC sera. Protein expression of NRP-1 was analyzed by Western blotting, cell proliferation by 5′-bromodeoxyuridine assay, migration capacity by wound-healing assay, and capillary-like tube formation by Matrigel assay.Results.Serum levels of pan-VEGF were increased in patients with VEDOSS and SSc versus HC (p = 0.05 and p = 0.003, respectively). Serum levels of sNRP-1 were significantly reduced in patients with VEDOSS and SSc compared with controls (p = 0.012 and p = 0.027, respectively). NRP-1 expression was decreased in H-MVEC stimulated with VEDOSS sera (p < 0.001 vs HC). Proliferation was reduced in H-MVEC stimulated either with VEDOSS or SSc sera in comparison with HC sera (p = 0.015 and p = 0.043, respectively). Wound healing was compromised in H-MVEC stimulated with VEDOSS and SSc sera versus HC sera (p < 0.01 for both). Capillarogenesis was decreased in H-MVEC stimulated with VEDOSS sera (p < 0.01) and SSc sera (p < 0.001) compared with cells stimulated with HC sera.Conclusion.Similar to patients with SSc, patients with VEDOSS already present biological signs of endothelial dysfunction. Our data demonstrate that VEDOSS sera significantly modify endothelial cell behavior and impair the angiogenic potential of the microvascular system.

scholarly journals Co-transfection with BMP2 and FGF2 via chitosan nanoparticles potentiates osteogenesis in human adipose-derived stromal cells in vitro

2021 ◽  
Vol 49 (3) ◽  
pp. 030006052199767
Author(s):  
Ying Hu ◽  
Qing-Wei Zhao ◽  
Zheng-Cai Wang ◽  
Qing-Qing Fang ◽  
He Zhu ◽  
...  
Keyword(s):  
Real Time ◽  
Chitosan Nanoparticles ◽  
Human Adipose Tissue ◽  
Bone Morphogenetic Protein 2 ◽  
Expression Vectors ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Mrna Levels ◽  
Gene And Protein Expression

Objective To investigate if co-transfection of human bone morphogenetic protein 2 (BMP-2, BMP2) and human fibroblast growth factor 2 (FGF2, FGF2) via chitosan nanoparticles promotes osteogenesis in human adipose tissue-derived stem cells (ADSCs) in vitro. Materials and Methods Recombinant BMP2 and/or FGF2 expression vectors were constructed and packaged into chitosan nanoparticles. The chitosan nanoparticles were characterized by atomic force microscopy. Gene and protein expression levels of BMP-2 and FGF2 in ADSCs in vitro were evaluated by real-time polymerase chain reaction (PCR), western blot, and enzyme-linked immunosorbent assay. Osteocalcin (OCN) and bone sialoprotein (BSP) gene expression were also evaluated by real-time PCR to assess osteogenesis. Results The prepared chitosan nanoparticles were spherical with a relatively homogenous size distribution. The BMP2 and FGF2 vectors were successfully transfected into ADSCs. BMP-2 and FGF2 mRNA and protein levels were significantly up-regulated in the co-transfection group compared with the control group. OCN and BSP mRNA levels were also significantly increased in the co-transfection group compared with cells transfected with BMP2 or FGF2 alone, suggesting that co-transfection significantly enhanced osteogenesis. Conclusions Co-transfection of human ADSCs with BMP2/FGF2 via chitosan nanoparticles efficiently promotes the osteogenic properties of ADSCs in vitro.

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