Wound fluid under occlusive dressings from diabetic patients show an increased angiogenic response and fibroblast migration

Abstract Background Peripheral artery disease (PAD) is highly prevalent in people with type 2 diabetes and associates with chronic limb ischemia and poor prognosis. Understanding the mechanisms of impaired blood vessel growth in diabetic patients is of paramount importance to develop new angiogenic therapies in this setting. Dysregulation of epigenetic mechanisms of gene transcription in vascular cells contributes to cardiovascular disease development but is currently not targeted by therapies. Apabetalone (RVX-208) – an FDA approved small molecule inhibitor of the epigenetic readers bromodomain and extra-terminal (BET) proteins – has recently shown to modulate transcriptional programs implicated in vascular inflammation and atherosclerosis. Purpose To investigate RVX-208 effects in modulating angiogenic response and post-ischemic vascularization in diabetes. Methods Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 20 mM) for 48 hours in presence of RVX-208 (20μM) or vehicle (DMSO). Scratch and tube formation assays were performed to investigate the impact of RVX-208 on angiogenic properties of HAECs. T1D mice (streptozotocin-induced diabetes) and T2D mice (Lepdb/db) were orally treated with apabetalone or vehicle for 5 days. Hindlimb ischemia was induced in T1D mice & blood flow recovery analysed at 30 minutes, 7 and 14 days by laser Doppler imaging. Sprouting and matrigel plug assays were performed in Lepdb/db mice. Gastrocnemius muscle samples from patients with and without T2D were employed to translate our experimental findings. Results HG impaired HAECs migration and tube formation as compared to NG, whereas treatment with RVX-208 rescued HG-induced impairment of angiogenic properties. Real time PCR arrays in HG-treated HAECs showed that RVX-208 treatment prevents the dysregulation of genes implicated in endothelial migration, sprouting and inflammation, namely the anti-angiogenic molecule thrombospondin (THBS1), VEGF-A, IL-1β, IL-6, VCAM-1, and CXCL1. Of interest, both gene silencing of BET protein (BRD4) or its pharmacological inhibition by RVX-208 reduced THBS1 expression while restoring VEGFA levels in HG-treated HAECs. ChIP assays showed the enrichment of both BRD4 and the active chromatin mark H3K27Ac on THBS1 promoter. Mechanistic experiments uncovered the inhibitory role of THBS1 on VEGFA signalling, as also confirmed by STRING analysis. Treatment of T1D mice with RVX-208 improved blood flow reperfusion and vascular density at 14 days as compared to vehicle-treated animals. Moreover, RVX-208 restored endothelial sprouting in T2D-Lepdb/db mice. Of clinical relevance, THBS1 was upregulated while VEGFA expression was reduced in gastrocnemius muscle specimens from T2D patients with PAD as compared to non-diabetic controls. Conclusion In vivo targeting of BET-proteins by RVX-208 may represents a novel therapeutic approach to boost post-ischemic neovascularization in diabetes. FUNDunding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of Zurich

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The Host Defence Peptide LL-37 is Susceptible to Proteolytic Degradation by Wound Fluid Isolated from Foot Ulcers of Diabetic Patients

International Journal of Peptide Research and Therapeutics ◽

10.1007/s10989-014-9410-3 ◽

2014 ◽

Vol 20 (4) ◽

pp. 457-464 ◽

Cited By ~ 13

Author(s):

Maelíosa T. C. McCrudden ◽

Denise T. F. McLean ◽

Mei Zhou ◽

Julia Shaw ◽

Gerard J. Linden ◽

...

Keyword(s):

Host Defence ◽

Foot Ulcers ◽

Proteolytic Degradation ◽

Diabetic Patients ◽

Wound Fluid

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Pharmacological blockade of histone methyltransferase SETD7 restores angiogenic response in experimental diabetes

European Heart Journal ◽

10.1093/eurheartj/ehab724.3203 ◽

2021 ◽

Vol 42 (Supplement_1) ◽

Author(s):

S Mohammed ◽

S C Costantino ◽

A A Akhmedov ◽

G K Karsay ◽

S A Ambrosini ◽

...

Keyword(s):

Blood Flow ◽

Blood Vessel ◽

Experimental Diabetes ◽

Tube Formation ◽

Diabetic Patients ◽

Rna Seq ◽

Angiogenic Response ◽

Histone 3 ◽

Vessel Growth ◽

Pharmacological Blockade

Abstract Background Peripheral artery disease (PAD) is highly prevalent in patients with diabetes and associates with a high rate of limb amputation and poor prognosis. Surgical and catheter-based revascularization have failed to improve outcome in diabetic patients with PAD. Hence, a need exists to develop new treatment strategies able to promote blood vessel growth in the ischemic limb of diabetic patients. Mono-methylation of histone 3 at lysine 4 (H3K4me1) - a specific epigenetic signature induced by the methyltransferase SETD7 - favours a chromatin active and open state thus enabling the gene transcription. Purpose To investigate whether SETD7-dependent epigenetic changes modulate post-ischemic vascularization in experimental diabetes. Methodology Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 20 mM) concentrations for 48 hours. Unbiased gene expression profiling was performed by RNA sequencing (RNA-seq) followed by Ingenuity Pathway Analysis (IPA). In vitro angiogenic assays like migration assay & tube formation assay were performed. Pharmacological blockade of SETD7 was achieved by using the highly selective inhibitor called (R)-PFI-2. T1D mice (streptozotocin-induced diabetes) was orally treated with (R)-PFI-2 and with vehicle for 21 days and followed by induction of hindlimb ischemia. Blood flow recovery was analyzed at 30 minutes, 7 and 14 days by laser doppler imaging. Gastrocnemius muscle samples from patients with and without T2D were employed to translate our experimental findings. Results RNA-seq in HG-treated HAECs revealed a profound upregulation of the methyltransferase SETD7, an enzyme involved in mono-methylation of lysine 4 at histone 3 (H3K4me1). SETD7 upregulation in HG-treated HAECs was associated with an increase of H3K4-mono-methylation levels as well as with impaired endothelial cell migration and tube formation. Of interest, both gene silencing (SETD7-siRNA) and pharmacological blockade of SETD7 by (R)-PFI-2 rescued hyperglycemia-induced impairment of angiogenic properties in HAECs. RNA-seq in HG-treated HAECs with and without SETD7 depletion unveiled an array of differentially expressed genes, which were mainly involved in blood vessel growth and angiogenic response. Among dysregulated genes, Chromatin immunoprecipitation (ChIP) assays showed that SETD7 specifically mono-methylates H3K4m1 in proximity of Semaphorin-3G (SEMA3G) promoter, thus regulating its expression. Treatment of T1D mice with (R)-PFI-2 improved blood flow reperfusion at 14 days as compared to vehicle-treated animals. Finally, SETD7/SEMA3G axis was upregulated in muscle specimens from T2D patients. Conclusion Targeting SETD7 represents a novel epigenetic-based therapy to boost neovascularization in diabetic patients with PAD. FUNDunding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of Zurich

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IL-1β Impaired Diabetic Wound Healing by Regulating MMP-2 and MMP-9 through the p38 Pathway

Mediators of Inflammation ◽

10.1155/2021/6645766 ◽

2021 ◽

Vol 2021 ◽

pp. 1-10

Author(s):

Jiezhi Dai ◽

Junjie Shen ◽

Yimin Chai ◽

Hua Chen

Keyword(s):

Diabetes Mellitus ◽

Cell Proliferation ◽

Wound Healing ◽

Human Fibroblasts ◽

Diabetic Patients ◽

Diabetic Wound ◽

Fibroblast Migration ◽

Cell Proliferation And Migration ◽

And Migration ◽

Proliferation And Migration

Diabetes mellitus is one of the most prominent metabolic disorders in the world, and insulin resistance in diabetic patients leads to several complications including increased inflammation and delayed wound healing. Fibroblast migration and reepithelialization play a significant role in wound healing. In this study, we explored the effects of IL-1β signaling on proliferation and migration of human fibroblasts from diabetic wound tissues. We observed elevated levels of IL-1β in samples from diabetic patients when compared to normal wound tissues. At high concentrations, IL-1β inhibited cell proliferation and migration in ex vivo fibroblast cultures. Moreover, expression of matrix metalloproteinases (MMPs) was upregulated, and tissue inhibitor of metalloproteinases (TIMPs) was downregulated in diabetic wound tissues and cells. These effects were regulated by levels of IL-1β. Furthermore, IL-1β induced p38 phosphorylation thereby activating the p38 MAPK pathway that in turn regulated the expression of MMPs and TIMPs. Together, our study identifies a novel mechanism behind delayed wound closure in diabetes mellitus that involves IL-1β-dependent regulation of cell proliferation and migration.

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Acute and Chronic Wound Fluid Inversely Influence Wound Healing in an in-Vitro 3D Wound Model

Journal of Tissue Repair and Regeneration ◽

10.14302/issn.2640-6403.jtrr-17-1818 ◽

2017 ◽

Vol 1 (1) ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

Besser Manuela ◽

Khosravani Milad ◽

Severing Anna-Lena ◽

Rembe Julian-Dario ◽

Stuermer Ewa Klara

Keyword(s):

Cell Proliferation ◽

Chronic Wounds ◽

Healing Process ◽

Collagen Matrix ◽

Wound Fluid ◽

Fibroblast Migration ◽

And Migration ◽

The Impact

If a wound progressively heals or the healing process is impaired is basically influenced by the surrounding milieu. This is reflected by the wound fluid. Its specific composition triggers the migration, proliferation and differentiation of dermal and epidermal cells which so far was not sufficiently examined in 2D cell culture models. The influence of the different wound entities was analyzed on a newly implemented three dimensional in-vitro model, which improved the transferability to the in-vivo situation. The influence of pooled wound fluids from patients suffering from acute or chronic wounds were investigated within a time period of 10 days after wound application. Histological and immunohistochemical analyses were performed addressing the impact of AWF and CWF on regeneration, such as cell proliferation, fibroblast activity and cell migration. AWF slightly stimulated fibroblast migration while CWF inhibited their activation and migration. The CXCR4- immunopositive population was continuously decreased compared to the control and AWF treatment. The expression of FAP was enhanced under AWF and medium. In keratinocytes CWF massively stimulated cell proliferation initiating on day six after injury. The presence of 10% CWF inhibited fibroblast activation and migration and induced the degradation of the collagen matrix. Keratinocytes were stimulated to proliferate, resulting in healing inhibiting hyperplasia. Transferred to human wounds, no effective wound closure would be achieved because of the de-regulation of pro-proliferative and migration-stimulating factors and a degraded extracellular matrix. This newly implemented 3D study model represents a novel appropriate in-vitro system for studying healing mechanisms and potential therapeutic applications.

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Intramuscular Nerve and Neuromuscular Junction Alteration In Extraocular Muscles of Diabetic Mice

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100120102 ◽

1985 ◽

Vol 43 ◽

pp. 682-683

Author(s):

Bruce R. Pachter

Keyword(s):

Diabetes Mellitus ◽

Peripheral Nerves ◽

Sudden Onset ◽

Extraocular Muscles ◽

Diabetic Patients ◽

Oculomotor Palsy ◽

Third Nerve Palsy ◽

Patients With Diabetes ◽

Intramuscular Nerve ◽

Oculomotor Nerves

Diabetes mellitus is one of the commonest causes of neuropathy. Diabetic neuropathy is a heterogeneous group of neuropathic disorders to which patients with diabetes mellitus are susceptible; more than one kind of neuropathy can frequently occur in the same individual. Abnormalities are also known to occur in nearly every anatomic subdivision of the eye in diabetic patients. Oculomotor palsy appears to be common in diabetes mellitus for their occurrence in isolation to suggest diabetes. Nerves to the external ocular muscles are most commonly affected, particularly the oculomotor or third cranial nerve. The third nerve palsy of diabetes is characteristic, being of sudden onset, accompanied by orbital and retro-orbital pain, often associated with complete involvement of the external ocular muscles innervated by the nerve. While the human and experimental animal literature is replete with studies on the peripheral nerves in diabetes mellitus, there is but a paucity of reported studies dealing with the oculomotor nerves and their associated extraocular muscles (EOMs).

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A Comparison Between Sterological Point Counting and Digitizing Tablets

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100120023 ◽

1985 ◽

Vol 43 ◽

pp. 666-667

Author(s):

John M. Basgen ◽

Eileen N. Ellis ◽

S. Michael Mauer ◽

Michael W. Steffes

Keyword(s):

Electron Microscopy ◽

Kidney Tissue ◽

Volume Density ◽

Diabetic Patients ◽

Normal Kidney ◽

Point Counting ◽

Normal Kidney Tissue ◽

Biopsy Specimens ◽

Mitochondrial Volume ◽

Kidney Lesions

To determine the efficiency of methods of quantitation of the volume density of components within kidney biopsies, techniques involving a semi-automatic digitizing tablet and stereological point counting were compared.Volume density (Vv) is a parameter reflecting the volume of a component to the volume that contains the component, e.g., the fraction of cell volume that is made up of mitochondrial volume. The units of Vv are μm3 /μm3.Kidney biopsies from 15 patients were used. Five were donor biopsies performed at the time of kidney transplantation (patients 1-5, TABLE 1) and were considered normal kidney tissue. The remaining biopsies were obtained from diabetic patients with a spectrum of diabetic kidney lesions. The biopsy specimens were fixed and embedded according to routine electron microscogy protocols. Three glomeruli from each patient were selected randomly for electron microscopy. An average of 12 unbiased and systematic micrographs were obtained from each glomerulus and printed at a final magnification of x18,000.

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Biological and biomedical applications of collagenous biomaterials

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100161849 ◽

1990 ◽

Vol 48 (3) ◽

pp. 856-857

Author(s):

Yasushi P. Kato ◽

Michael G. Dunn ◽

Frederick H. Silver ◽

Arthur J. Wasserman

Keyword(s):

Biomedical Applications ◽

Soft Tissues ◽

Collagen Fibers ◽

Bone Collagen ◽

Cover Slip ◽

Fibroblast Migration ◽

Cellular Expression ◽

Defect Repair

Collagenous biomaterials have been used for growing cells in vitro as well as for augmentation and replacement of hard and soft tissues. The substratum used for culturing cells is implicated in the modulation of phenotypic cellular expression, cellular orientation and adhesion. Collagen may have a strong influence on these cellular parameters when used as a substrate in vitro. Clinically, collagen has many applications to wound healing including, skin and bone substitution, tendon, ligament, and nerve replacement. In this report we demonstrate two uses of collagen. First as a fiber to support fibroblast growth in vitro, and second as a demineralized bone/collagen sponge for radial bone defect repair in vivo.For the in vitro study, collagen fibers were prepared as described previously. Primary rat tendon fibroblasts (1° RTF) were isolated and cultured for 5 days on 1 X 15 mm sterile cover slips. Six to seven collagen fibers, were glued parallel to each other onto a circular cover slip (D=18mm) and the 1 X 15mm cover slip populated with 1° RTF was placed at the center perpendicular to the collagen fibers. Fibroblast migration from the 1 x 15mm cover slip onto and along the collagen fibers was measured daily using a phase contrast microscope (Olympus CK-2) with a calibrated eyepiece. Migratory rates for fibroblasts were determined from 36 fibers over 4 days.

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