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3 pages, 168 KiB

Open AccessEditorial

Arteriogenesis and Therapeutic Angiogenesis in Its Multiple Aspects

by Elisabeth Deindl and Paul H. A. Quax

Cells 2020, 9(6), 1439; https://doi.org/10.3390/cells9061439 - 10 Jun 2020

Cited by 9 | Viewed by 2458

Abstract

Arteriogenesis, also frequently called collateral formation or even therapeutic angiogenesis, comprises those processes that lead to the formation and growth of collateral blood vessels that can act as natural bypasses to restore blood flow to distal tissues in occluded arteries [...] Full article

(This article belongs to the Special Issue Arteriogenesis and Therapeutic Neovascularization)

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11 pages, 5551 KiB

Open AccessArticle

Control of Angiogenesis via a VHL/miR-212/132 Axis

by Zhiyong Lei, Timothy D. Klasson, Maarten M. Brandt, Glenn van de Hoek, Ive Logister, Caroline Cheng, Pieter A. Doevendans, Joost P. G. Sluijter and Rachel H. Giles

Cells 2020, 9(4), 1017; https://doi.org/10.3390/cells9041017 - 19 Apr 2020

Cited by 12 | Viewed by 4045

Abstract

A common feature of tumorigenesis is the upregulation of angiogenesis pathways in order to supply nutrients via the blood for the growing tumor. Understanding how cells promote angiogenesis and how to control these processes pharmaceutically are of great clinical interest. Clear cell renal [...] Read more.

A common feature of tumorigenesis is the upregulation of angiogenesis pathways in order to supply nutrients via the blood for the growing tumor. Understanding how cells promote angiogenesis and how to control these processes pharmaceutically are of great clinical interest. Clear cell renal cell carcinoma (ccRCC) is the most common form of sporadic and inherited kidney cancer which is associated with excess neovascularization. ccRCC is highly associated with biallelic mutations in the von Hippel–Lindau (VHL) tumor suppressor gene. Although upregulation of the miR-212/132 family and disturbed VHL signaling have both been linked with angiogenesis, no evidence of a possible connection between the two has yet been made. We show that miRNA-212/132 levels are increased after loss of functional pVHL, the protein product of the VHL gene, in vivo and in vitro. Furthermore, we show that blocking miRNA-212/132 with anti-miRs can significantly alleviate the excessive vascular branching phenotype characteristic of vhl^−/− mutant zebrafish. Moreover, using human umbilical vascular endothelial cells (HUVECs) and an endothelial cell/pericyte coculture system, we observed that VHL knockdown promotes endothelial cells neovascularization capacity in vitro, an effect which can be inhibited by anti-miR-212/132 treatment. Taken together, our results demonstrate an important role for miRNA-212/132 in angiogenesis induced by loss of VHL. Intriguingly, this also presents a possibility for the pharmaceutical manipulation of angiogenesis by modulating levels of MiR212/132. Full article

(This article belongs to the Special Issue Arteriogenesis and Therapeutic Neovascularization)

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Figure 1

Figure 1
Characterization of miR-132 expression under hypoxic and pseudo-hypoxic conditions: (A) The expression of miR-132 in human umbilical vascular endothelial cells (HUVECs) under normoxia and hypoxia as compared by qPCR. (B) The expression of von Hippel–Lindau (VHL) in HUVECs after transfection with siRNA against VHL and the expression of miR-132 in siSham and siVHL transfected HUVECs as compared by qPCR. (C) The expression of miR-132 in wildtype (WT) and vhl−/− mutant zebrafish as compared by qPCR. (D) The expression of known miR-132 target PTEN (phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase) in HUVECs treated with miR-132/212 mimics versus control as compared by qPCR. (E) The expression of miR-132 in established VHL−/− lines RCC10, A498, and 786-0 as well as the same lines reconstituted with ectopic VHL. The presented data is a mean of 3 in-depended PCR experiments with counting error. (F) Relative expression of miR-132 and 212 in different tissues in mouse. Note miR-132 expression is considerably higher than miR-212. n = 3. (G) The expression of miR-132 in healthy kidney tissue and ccRCC from two patients with known bilateral VHL mutations in their tumor as shown by miR-132 in situ hybridization. miR-132 in situ is in purple blue. Light eosin counterstaining appears pink. * p < 0.05; ** p < 0.01; *** p < 0.001 Full article ">Figure 2
Reduced levels of VHL enhances endothelial cell neovascularization capacity and can be inhibited by blocking miR-132 or miR-212. (A) Schematic outline of the coculture experiment with HUVECs and pericytes. (B) Representative images showing the analysis process of tubular structures in the endothelial cells and pericytes coculture assay. (C) VHL siRNA knockdown in HUVECs enhances endothelial cell neovascularization capacity. (D) Blocking miR-132/212 inhibits neovascularization enhancement induced by VHL knockdown. Cell images are used to produce skeletonized 2D images which can be analyzed automatically. * p < 0.05; ** p < 0.01; *** p < 0.001 Full article ">Figure 3
Inhibition of miR-132 or miR-212 suppresses VHL loss of function-induced vasculature outgrowth in zebrafish. (A) Schematic outline of the zebrafish embryo microinjection experiment. microRNA mimics and anti-miRs are injected into the yolk of the eggs on day 0 and imaged with a confocal microscope on day 5. (B) Schematic cartoon showing the area of the zebrafish embryo that is imaged after microinjection. The cloaca is marked with a red arrow. The imaging area is shown with a red box. The vessels of the tail are shown in green. (C) Representative images of zebrafish tail vascular structures in vhl+/− and vhl−/− zebrafish after injection with scrambled or miR-132 and miR-212 inhibitors. White arrows designate examples of structures which have been scored as branches. (D) Quantification of vascular branching in zebrafish tail structures after injection with scrambled control inhibitors, miR-132 inhibitors, or miR-212 inhibitors. (E) The expression levels of ptena and ptenb in WT and vhl−/− zebrafish determined by qPCR. * p < 0.05; ** p < 0.01. Full article ">Figure 4
Proposed mechanism of miR-132/212 in modulation of the VHL/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/ Protein kinase B(AKT)pathways. (A) During normoxia, hypoxia-inducible transcription factor 1 (HIF1) is ubiquitinated by the VHL-ubiquinition complex, targeting it for degradation. Some effactors, such as PTEN, antagonizes PI3k to prevent AKT from being activated. (B) Upon hypoxia, HIF1 can no longer be hydroxylated, which prohibits VHL-regulated degradation, and allows stabilized HIF1 to translocate to the nucleus, upregulating its downstream targets such as vascular endothelial growth factor (VEGF). VEGF in turn activates the PI3k-AKT pathway and upregulates miR-132/212 expression as well. Upregulated miR-132/212 inhibits effector (e.g., PTEN) expression, which in turn prolongs AKT activity. (C) VHL loss-of-function phenocopies hypoxic conditions even in the presence of oxygen (pseudo-hypoxia). Full article ">

14 pages, 3806 KiB

Open AccessArticle

The Lipopeptide MALP-2 Promotes Collateral Growth

by Kerstin Troidl, Christian Schubert, Ann-Kathrin Vlacil, Ramesh Chennupati, Sören Koch, Jutta Schütt, Raghav Oberoi, Wolfgang Schaper, Thomas Schmitz-Rixen, Bernhard Schieffer and Karsten Grote

Cells 2020, 9(4), 997; https://doi.org/10.3390/cells9040997 - 16 Apr 2020

Cited by 11 | Viewed by 5415

Abstract

Beyond their role in pathogen recognition and the initiation of immune defense, Toll-like receptors (TLRs) are known to be involved in various vascular processes in health and disease. We investigated the potential of the lipopeptide and TLR2/6 ligand macrophage activating protein of 2-kDA [...] Read more.

Beyond their role in pathogen recognition and the initiation of immune defense, Toll-like receptors (TLRs) are known to be involved in various vascular processes in health and disease. We investigated the potential of the lipopeptide and TLR2/6 ligand macrophage activating protein of 2-kDA (MALP-2) to promote blood flow recovery in mice. Hypercholesterolemic apolipoprotein E (Apoe)-deficient mice were subjected to microsurgical ligation of the femoral artery. MALP-2 significantly improved blood flow recovery at early time points (three and seven days), as assessed by repeated laser speckle imaging, and increased the growth of pre-existing collateral arteries in the upper hind limb, along with intimal endothelial cell proliferation in the collateral wall and pericollateral macrophage accumulation. In addition, MALP-2 increased capillary density in the lower hind limb. MALP-2 enhanced endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) release from endothelial cells and improved the experimental vasorelaxation of mesenteric arteries ex vivo. In vitro, MALP-2 led to the up-regulated expression of major endothelial adhesion molecules as well as their leukocyte integrin receptors and consequently enhanced the endothelial adhesion of leukocytes. Using the experimental approach of femoral artery ligation (FAL), we achieved promising results with MALP-2 to promote peripheral blood flow recovery by collateral artery growth. Full article

(This article belongs to the Special Issue Arteriogenesis and Therapeutic Neovascularization)

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Figure 1

Figure 1
MALP-2 improved the perfusion recovery and collateral growth in the hind limb following femoral artery ligation (FAL) in hypercholesterolemic Apoe-deficient mice. (a) Following the FAL, the perfusion recovery was determined by laser Speckle perfusion imaging for C57BL/6, BALB/c and hypercholesterolemic Apoe-KO mice (12 weeks on a high fat diet (HFD)) treated with MALP-2 or PBS (control) pre/post the FAL, after three and seven days and, in Apoe-KO mice, after 10 days. Data are expressed as the ratio of the ligated and the non-ligated hind limb. ** P < 0.01, N = 4–7. (b) Representative laser speckle perfusion images indicate the effect of MALP-2 compared to the control (PBS) on perfusion recovery in the ligated hind limbs of Apoe-KO mice pre/post the FAL and after 3, 7 and 10 days. (c) Representative haematoxilin-eosin staining of cross sections of collateral arteries in the adductor muscle of the ligated and the non-ligated hind limbs of hypercholesterolemic Apoe-KO mice treated with MALP-2 or PBS (control) 10 days after the FAL and the corresponding morphometric analysis of the collateral diameter and wall area. Scale bar = 10 µm. * P < 0.05 vs. control, N = 6–14 collaterals. Full article ">Figure 2
MALP-2 increased pericollateral macrophage accumulation, endothelial cell proliferation and downstream angiogenesis following FAL. This shows the representative immunostaining of cross sections of collateral arteries in the adductor muscle and the calf muscle of the ligated hind limb in hypercholesterolemic Apoe-KO mice treated with MALP-2 and PBS (control) 3, 7 and 10 days after the FAL and the corresponding quantitative analysis. (a) CD68 staining to assess the accumulation of macrophages around the collateral (α-SMA indicates the media of the collateral wall). Scale bar = 25 µm. (b) Ki67 staining to determine the portion of proliferating CD31-positive collateral endothelial cells (white arrow heads). Scale bar = 25 µm. (c) CD31 indicates capillary density in the calf muscle. Scale bar = 50 µm. * P <0.05, ** P <0.01 vs. control, N = up to 20 collaterals, n.d. = not detected. Full article ">Figure 3
MALP-2 up-regulated inflammatory genes in the upper hind limb muscle. Tissue pieces of the adductor muscles of C57BL/6 mice were isolated and stimulated ex vivo with MALP-2 (1 µg/mL); Ccl2, Gm-csf, Il-1β and Tnf-α mRNA levels were analyzed after the indicated times by (a) real-time PCR and (b) the corresponding protein in the supernatant after 6 h by ELISA. CXCL12 mRNA levels were analyzed (c) in tissue pieces of the adductor muscle of C57BL/6 mice ex vivo and in (d) MyEND cells following MALP-2 stimulation (1 µg/mL) after the indicated times by real-time PCR. * P < 0.05, ** P < 0.01 vs. control, N = 4–6. Full article ">Figure 4
MALP-2 improved NO-dependent vascular relaxation in the mesenteric arteries of C57BL/6 mice. (a) The relaxation response to acetylcholine (ACh 0.001–10 µM) during phenylephrine-induced (PE, 10 µM) contraction in mesenteric arteries incubated with MALP-2 or PBS (control), N = 6. (b) The relaxation response to ACh (0.01–10 µM) during K+-induced (60 mM) contraction in mesenteric arteries incubated with indomethacin (10 µM, COX-inhibitor) and MALP-2 or PBS, N = 3. (c) The relaxation response to ACh (0.001–10 µM) in the presence of L-NAME (100 µM, NOS inhibitor) and indomethacin (10 µM). A.U.C. = area under the curve, * P < 0.05 vs. control, N = 3. Full article ">Figure 5
MALP-2 enhanced the endothelial cell-derived NO release. MyEnd cells were stimulated with MALP-2 (1 µg/mL); (a) the AKT phosphorylation (p-AKT) as well as (b) the eNOS phosphorylation (p-eNOS) were analyzed after the indicated times by Western blot and (c) the NO release was analyzed with the Griess reagent. The numbers between panels indicate fold-change vs. unstimulated after normalization to total AKT or eNOS, respectively. β-Actin was used as the loading control. * P < 0.05 vs. control, N = 4–5. Full article ">Figure 6
MALP-2 up-regulated endothelial adhesion molecules and enhanced the endothelial adhesion of monocytic cells. (a) The MyEnd cells were stimulated with MALP-2 (1 µg/mL) and the VCAM-1, ICAM-1, E-selectin and P-selectin mRNA levels were analyzed after the indicated times by real-time PCR. * P < 0.05, ** P < 0.01 vs. control, N = 6–8. (b) The MyEnd cells were stimulated with MALP-2 (1 µg/mL) and the VCAM-1 protein expression was analyzed after the indicated times by Western blot. β-Actin was used as the loading control. The numbers between panels indicate fold-change vs. unstimulated after normalization to β-Actin. * P < 0.05 vs. control, N = 4–5. (c) Fluorescence images depicting calcein-AM-labeled J774A.1 cells on a MyEnd monolayer with or without pretreatment with MALP-2 (1 µg/mL) for 6 h with an additional adhesion time of 1 h and the corresponding quantitative analysis. Pictures before and after washing are shown. Scale bar = 100 µm, ** P < 0.01 vs. control, N = 3. Full article ">

15 pages, 5668 KiB

Open AccessFeature PaperArticle

Contribution of the Potassium Channels K_V1.3 and K_Ca3.1 to Smooth Muscle Cell Proliferation in Growing Collateral Arteries

by Manuel Lasch, Amelia Caballero Martinez, Konda Kumaraswami, Hellen Ishikawa-Ankerhold, Sarah Meister and Elisabeth Deindl

Cells 2020, 9(4), 913; https://doi.org/10.3390/cells9040913 - 8 Apr 2020

Cited by 11 | Viewed by 3226

Abstract

Collateral artery growth (arteriogenesis) involves the proliferation of vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Whereas the proliferation of ECs is directly related to shear stress, the driving force for arteriogenesis, little is known about the mechanisms of SMC proliferation. Here [...] Read more.

Collateral artery growth (arteriogenesis) involves the proliferation of vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Whereas the proliferation of ECs is directly related to shear stress, the driving force for arteriogenesis, little is known about the mechanisms of SMC proliferation. Here we investigated the functional relevance of the potassium channels K_V1.3 and K_Ca3.1 for SMC proliferation in arteriogenesis. Employing a murine hindlimb model of arteriogenesis, we found that blocking K_V1.3 with PAP-1 or K_Ca3.1. with TRAM-34, both interfered with reperfusion recovery after femoral artery ligation as shown by Laser-Doppler Imaging. However, only treatment with PAP-1 resulted in a reduced SMC proliferation. qRT-PCR results revealed an impaired downregulation of α smooth muscle-actin (αSM-actin) and a repressed expression of fibroblast growth factor receptor 1 (Fgfr1) and platelet derived growth factor receptor b (Pdgfrb) in growing collaterals in vivo and in primary murine arterial SMCs in vitro under K_V1.3. blockade, but not when K_Ca3.1 was blocked. Moreover, treatment with PAP-1 impaired the mRNA expression of the cell cycle regulator early growth response-1 (Egr1) in vivo and in vitro. Together, these data indicate that K_V1.3 but not K_Ca3.1 contributes to SMC proliferation in arteriogenesis. Full article

(This article belongs to the Special Issue Arteriogenesis and Therapeutic Neovascularization)

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Figure 1

Figure 1
Photographs of superficial collateral arteries in mouse adductor muscles. Photographs were taken 7 days after induction of arteriogenesis by femoral artery ligation (left picture) or sham operation (right picture). Mice were perfused with latex to better visualize collateral arteries. Pre-existing collaterals appear very fine and straight (arrows, right picture). Seven days after induction of arteriogenesis, grown collateral arteries show a typical corkscrew formation with increased vascular caliber size (arrows, left picture). Scale bar 5 mm Full article ">Figure 2
Localization of KCa3.1 in ECs and SMCs of murine collateral arteries. (a) Representative confocal immunofluorescence images of transversal sections of collateral arteries isolated 3 h after induction of arteriogenesis. Tissue sections were stained with an antibody against KCa3.1 (green), together with the SMC marker αSM-actin (red), the EC marker CD31 (grey), and DAPI (blue); (b,c) Scatterplots showing the colocalization analysis, (left lower panel) represents pixels that have low intensity levels in both channels, green and red (b), or green and gray (c). Quadrant 4 (lower left bottom) represents pixels that are referred to as background and are not taken into consideration for colocalization analysis. Quadrant 1 represents pixels that have high green intensities and low red intensities and Quadrant 2 represents pixels that have high red intensities and low green intensities. Quadrant 3 represents pixels with high intensity levels in both green and red (b) or green and gray (c). These pixels are considered to be colocalized. Bright field image is also displayed. (c) 3D projection surface rendering is showing the localization of the KCa3.1 with the labelling CD 31 and αSM-actin display on the panel (c) right lower position. Scale bar 20 µm. Full article ">Figure 3
Localization of KV1.3 in ECs and SMCs of murine collateral arteries. (a) Representative confocal immunofluorescence images of transversal sections of collateral arteries isolated 3 h after induction of arteriogenesis. Tissue sections were stained with an antibody against KV1.3 (green), together with the SMC marker αSM-actin (red), the EC marker CD31 (grey), and DAPI (blue); (b,c) Scatterplots showing the colocalization analysis. Quadrant 4 (left lower left panel) represents pixels that have low intensity levels in both channels, green and red (b) or green and grey (c), and these pixels are referred to as background and are not taken into consideration for colocalization analysis. Quadrant 1 represent pixels that have high green intensities and low red intensities and Quadrant 2 represents pixels that have high red intensities and low green intensities. Quadrant 3 represents pixels with high intensity levels in both green and red in (b) and green and grey in (c). These pixels are considered to be colocalized. Scale bar 20 µm. Bright field image is also displayed. (c) 3D projection surface rendering is showing the localization of the KV1.3 with the labelling CD 31 and αSM-actin on the panel (c) right lower position. Full article ">Figure 4
Laser Doppler perfusion measurements. Line plot (left panel) along with corresponding flux images (right panel) of laser Doppler perfusion measurements. Mice were treated with solvent (control), PAP-1, or TRAM-34, respectively, and the perfusion was calculated by right to left (occlusion (occ) to sham) ratio before, immediately after, and at day 3 and 7 after the surgical procedure (left panel). Data are means ± SEM, n = 6 per group. * p < 0.05 (PAP-1 vs. control) and # p < 0.05 (TRAM-34 vs. control) from two-way ANOVA with Bonferroni’s multiple comparison test. The right panel shows representative flux images of murine paws with the tail in the center. Cold colors (blue, green) indicate low perfusion, whereas warm colors (yellow, red) indicate high perfusion (see scale). Full article ">Figure 5
BrdU incorporation and αSM-actin expression in collaterals. (a,b) Bar graphs represent the results of quantitative analyses of BrdU+ ECs (left panels) and SMCs (right panels) in solvent (control), (a) PAP-1 or (b) TRAM-34-treated mice at day 7 after induction of arteriogenesis. Data are means ± SEM, n = 3 mice per group. * p < 0.05 from unpaired student´s t-test. The numbers of BrdU+ cells in control collaterals were defined as 100%; (c) Representative picture of a BrdU stained collateral at day 7 after induction of arteriogenesis. Scale bar 20 µm; (d,e) The bar graphs represent the expression levels of αSM-actin (occlusion/sham (occ/sham)) in collateral arteries (d) at different time points after induction of arteriogenesis or (e) at 12 h after induction of arteriogenesis in control, PAP-1, or TRAM-34 treated mice. The qRT-PCR results were normalized to the expression level of the 18SrRNA. Data are means ± SEM, n > 3 per group. * p < 0.05 from unpaired student’s t-test and refers in (d) to occ vs. sham. Full article ">Figure 6
Immunocytological analyses on KV1.3 and KCa3.1 localization in mouse primary artery SMCs. Cells were stained with antibodies against the KV1.3 (upper panels, green) or the KCa3.1 channel (middle panels, green) together with an antibody against the SMC marker αSM-actin (red) and counterstained with DAPI (blue) to show the nuclei. For negative control (lower panels) the primary antibody was omitted. Scale bar 40 µm. Full article ">Figure 7
Proliferation assay of mouse primary artery SMCs. Mouse primary artery SMCs were cultured with 10% FCS with or without treatment of different concentrations of the KV1.3 blocker PAP-1 or the KCa3.1 blocker TRAM-34. Cell proliferation was investigated by means of BrdU incorporation. Values are expressed as percentages of the positive control (+), i.e., mouse primary artery SMCs stimulated with 10% FCS. For the negative control (–), mouse primary artery SMCs cultured with 2% FCS. Data are means ± SEM, n > 6 per group. * p < 0.05 from one-way ANOVA with Bonferroni’s multiple comparison test. Full article ">Figure 8
The qRT-PCR results of the expression levels of Fgfr1, Pdgfrb, and Egr1 in vitro and during arteriogenesis in vivo. (a,c) Bar graphs represent the mRNA expression levels of Fgfr1, Pdgfrb, or Egr1 in vitro and (b,d) in vivo. In vitro mouse primary artery SMCs were cultured without (control) or with 1 μM PAP-1 or 100 nM TRAM-34, respectively. In vivo the expression level of Fgfr1, Pdgfrb, and Egr1 were investigated 12 h after induction of arteriogenesis in collateral arteries and are expressed as occlusion (occ) to sham ratio. All qRT-PCR results were normalized to the expression level of the corresponding 18S rRNA. Data are means ± SEM, n = 3 per group. * p < 0.05 from one-way ANOVA with Bonferroni’s multiple comparison test. Full article ">

13 pages, 4620 KiB

Open AccessArticle

Local Mast Cell Activation Promotes Neovascularization

by Ilze Bot, Daniël van der Velden, Merel Bouwman, Mara J. Kröner, Johan Kuiper, Paul H. A. Quax and Margreet R. de Vries

Cells 2020, 9(3), 701; https://doi.org/10.3390/cells9030701 - 12 Mar 2020

Cited by 18 | Viewed by 3560

Abstract

Mast cells have been associated with arteriogenesis and collateral formation. In advanced human atherosclerotic plaques, mast cells have been shown to colocalize with plaque neovessels, and mast cells have also been associated with tumor vascularization. Based on these associations, we hypothesize that mast [...] Read more.

Mast cells have been associated with arteriogenesis and collateral formation. In advanced human atherosclerotic plaques, mast cells have been shown to colocalize with plaque neovessels, and mast cells have also been associated with tumor vascularization. Based on these associations, we hypothesize that mast cells promote angiogenesis during ischemia. In human ischemic muscle tissue from patients with end-stage peripheral artery disease, we observed activated mast cells, predominantly located around capillaries. Also, in mouse ischemic muscles, mast cells were detected during the revascularization process and interestingly, mast cell activation status was enhanced up to 10 days after ischemia induction. To determine whether mast cells contribute to both arteriogenesis and angiogenesis, mast cells were locally activated immediately upon hind limb ischemia in C57Bl/6 mice. At day 9, we observed a 3-fold increase in activated mast cell numbers in the inguinal lymph nodes. This was accompanied by an increase in the amount of Ly6C^high inflammatory monocytes. Interestingly, local mast cell activation increased blood flow through the hind limb (46% at day 9) compared to that in non-activated control mice. Histological analysis of the muscle tissue revealed that mast cell activation did not affect the number of collaterals, but increased the collateral diameter, as well as the number of CD31⁺ capillaries. Together, these data illustrate that locally activated mast cell contribute to arteriogenesis and angiogenesis. Full article

(This article belongs to the Special Issue Arteriogenesis and Therapeutic Neovascularization)

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Figure 1

Figure 1
Mast cells in calf muscles of patients with peripheral artery disease. (A) Representative high-magnification image of a non-activated (left) and activated (right) mast cell, stained using a chloro-acetate esterase (CAE) staining and indicated by arrows. The non-activated mast cell shows the pink granula in the cytoplasm of the cell, whereas the pink granula are being released in the surroundings of the activated mast cell. (B) Quantification of the number of mast cells/mm2 tissue, and the percentage of activated mast cells observed (n = 15). (C) Overview of a chloro-acetate esterase (CAE) staining of muscle tissue showing mast cells in pink (indicate by arrows) in between muscle fibers. (D) Representative overview images of mast cells surrounding microvessels (indicated by *) in human calf muscle tissue. Full article ">Figure 2
Mast cells in hind limb muscle tissue during ischemia in mice. (A) Representative images of mast cells in pink (CAE staining, indicated by arrows) in mouse ischemic muscle tissue (upper panels) and in close proximity to blood vessels (lower panels, indicated by *). (B) Mast cell density and (C) the percentage of activated mast cells in the hind limb muscle during recovery after ischemia. p-values of < 0.001 in comparison to t0 are indicated by *** p-value of < 0.05 in comparison to t7 is indicated by #. Full article ">Figure 3
Perfusion of the ischemic hind limb in mast cell (MC) activated vs. control mice. Perfusion (ischemic/non-ischemic) as measured by Laser Doppler Perfusion Imaging (LDPI) from day 0 to day 28 in the long-term experiment (A) and in the short-term experiment from day 0 up to day 9 (B) after ligation of the femoral artery. * p-value of < 0.05 between the groups. Full article ">Figure 4
Activated mast cell numbers in muscle tissue of the ischemic hind limb. (A) The number of activated mast cells as measured by histology in the ischemic vs. non-ischemic adductor muscles of mice in which mast cells were activated compared to control mice at day 9 after the induction of ischemia. (B) Total mast cell numbers per mm2 of ischemic soleus muscle tissue. (C) Number of activated mast cells per mm2 of soleus muscle tissue at day 9 after ligation. (D) Representative overview images of the soleus muscles with a resting (control, left) and an activated mast cell (dinitrophenyl hapten, DNP, right) in high-magnification inserts. Full article ">Figure 5
Arteriogenesis upon mast cell activation in the ischemic hind limb. The average collateral surface area in ischemic as compared to non-ischemic adductor muscles of mice in which mast cells were activated compared to controls at (A) 28 days after ligation and (B) 9 days after ligation. (C) The average collateral surface area in the soleus muscle of mice in which mast cells were activated compared to controls. (D) The collateral area ratio between ischemic and non-ischemic soleus muscles in mast cell activated compared to control mice at nine days after femoral artery ligation. (E). Representative pictures of smooth muscle cell actin positive collaterals in orange and nuclei in blue (DAPI) in both a control muscle and a muscle in which mast cells were activated. * p < 0.05. Full article ">Figure 6
Activated mast cells induce angiogenesis in the ischemic hind limb. (A) CD31 staining of the ischemic soleus muscles shows the presence of CD31+ capillaries in control mice and in mice in which mast cells were activated. (B) Quantification of the number of CD31+ capillaries in non-ischemic and ischemic soleus muscle with (white bars) or without local (black bars) mast cell activation, measured at nine days after femoral artery ligation. *p < 0.05. Full article ">Figure 7
Inflammatory cell analysis in the ischemic hind limb at nine days after ischemia induction. (A) Total CD117+FcεRI+ mast cell numbers and (B) the number of CD63+ activated mast cells were measured in the inguinal lymph node (iLN) draining from the ischemic hind limb of mice in the DNP-group and the controls using flow cytometry. (C) The number of CD11b+Ly6Chigh neutrophils in the iLN. (D) The number of CD11b+Ly6Glow monocytes of which, (E) Ly6Clow/mid and (F) Ly6Chigh monocytes. (G) Percentage of neutrophils, (H) percentage of total monocytes, and (I) percentage of inflammatory monocytes within the total monocyte population were measured in the circulation of control mice vs. mice in which mast cells were activated. (J) The number of macrophages per microscopic field in the ischemic soleus muscles of mice in which mast cells were activated vs. controls at nine days after ligation. (K) Representative micrographs of macrophages in red (indicated by arrows) and aSMA+ collaterals in cyan and nuclei (DAPI) in blue. * p < 0.05. Full article ">

18 pages, 6255 KiB

Open AccessFeature PaperArticle

Prolonged Hyperoxygenation Treatment Improves Vein Graft Patency and Decreases Macrophage Content in Atherosclerotic Lesions in ApoE3*Leiden Mice

by Laura Parma, Hendrika A. B. Peters, Fabiana Baganha, Judith C. Sluimer, Margreet R. de Vries and Paul H.A. Quax

Cells 2020, 9(2), 336; https://doi.org/10.3390/cells9020336 - 1 Feb 2020

Cited by 9 | Viewed by 3303

Abstract

Unstable atherosclerotic plaques frequently show plaque angiogenesis which increases the chance of rupture and thrombus formation leading to infarctions. Hypoxia plays a role in angiogenesis and inflammation, two processes involved in the pathogenesis of atherosclerosis. We aim to study the effect of resolution [...] Read more.

Unstable atherosclerotic plaques frequently show plaque angiogenesis which increases the chance of rupture and thrombus formation leading to infarctions. Hypoxia plays a role in angiogenesis and inflammation, two processes involved in the pathogenesis of atherosclerosis. We aim to study the effect of resolution of hypoxia using carbogen gas (95% O₂, 5% CO₂) on the remodeling of vein graft accelerated atherosclerotic lesions in ApoE3*Leiden mice which harbor plaque angiogenesis. Single treatment resulted in a drastic decrease of intraplaque hypoxia, without affecting plaque composition. Daily treatment for three weeks resulted in 34.5% increase in vein graft patency and increased lumen size. However, after three weeks intraplaque hypoxia was comparable to the controls, as were the number of neovessels and the degree of intraplaque hemorrhage. To our surprise we found that three weeks of treatment triggered ROS accumulation and subsequent Hif1a induction, paralleled with a reduction in the macrophage content, pointing to an increase in lesion stability. Similar to what we observed in vivo, in vitro induction of ROS in bone marrow derived macrophages lead to increased Hif1a expression and extensive DNA damage and apoptosis. Our study demonstrates that carbogen treatment did improve vein graft patency and plaque stability and reduced intraplaque macrophage accumulation via ROS mediated DNA damage and apoptosis but failed to have long term effects on hypoxia and intraplaque angiogenesis. Full article

(This article belongs to the Special Issue Arteriogenesis and Therapeutic Neovascularization)

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Figure 1

17 pages, 3129 KiB

Open AccessArticle

Isoform-Specific Roles of ERK1 and ERK2 in Arteriogenesis

by Nicolas Ricard, Jiasheng Zhang, Zhen W. Zhuang and Michael Simons

Cells 2020, 9(1), 38; https://doi.org/10.3390/cells9010038 - 21 Dec 2019

Cited by 19 | Viewed by 3487

Abstract

Despite the clinical importance of arteriogenesis, this biological process is poorly understood. ERK1 and ERK2 are key components of a major intracellular signaling pathway activated by vascular endothelial growth (VEGF) and FGF2, growth factors critical to arteriogenesis. To investigate the specific role of [...] Read more.

Despite the clinical importance of arteriogenesis, this biological process is poorly understood. ERK1 and ERK2 are key components of a major intracellular signaling pathway activated by vascular endothelial growth (VEGF) and FGF2, growth factors critical to arteriogenesis. To investigate the specific role of each ERK isoform in arteriogenesis, we used mice with a global Erk1 knockout as well as Erk1 and Erk2 floxed mice to delete Erk1 or Erk2 in endothelial cells, macrophages, and smooth muscle cells. We found that ERK1 controls macrophage infiltration following an ischemic event. Loss of ERK1 in endothelial cells and macrophages induced an excessive macrophage infiltration leading to an increased but poorly functional arteriogenesis. Loss of ERK2 in endothelial cells leads to a decreased arteriogenesis due to decreased endothelial cell proliferation and a reduced eNOS expression. These findings show for the first time that isoform-specific roles of ERK1 and ERK2 in the control of arteriogenesis. Full article

(This article belongs to the Special Issue Arteriogenesis and Therapeutic Neovascularization)

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17 pages, 2805 KiB

Open AccessArticle

VEGF-A-Cleavage by FSAP and Inhibition of Neo-Vascularization

by Özgür Uslu, Joerg Herold and Sandip M. Kanse

Cells 2019, 8(11), 1396; https://doi.org/10.3390/cells8111396 - 6 Nov 2019

Cited by 7 | Viewed by 3659

Abstract

Alternative splicing leads to the secretion of multiple forms of vascular endothelial growth factor-A (VEGF-A) that differ in their activity profiles with respect to neovascularization. FSAP (factor VII activating protease) is the zymogen form of a plasma protease that is activated (FSAPa) upon [...] Read more.

Alternative splicing leads to the secretion of multiple forms of vascular endothelial growth factor-A (VEGF-A) that differ in their activity profiles with respect to neovascularization. FSAP (factor VII activating protease) is the zymogen form of a plasma protease that is activated (FSAPa) upon tissue injury via the release of histones. The purpose of the study was to determine if FSAPa regulates VEGF-A activity in vitro and in vivo. FSAP bound to VEGF₁₆₅, but not VEGF₁₂₁, and VEGF₁₆₅ was cleaved in its neuropilin/proteoglycan binding domain. VEGF₁₆₅ cleavage did not alter its binding to VEGF receptors but diminished its binding to neuropilin. The stimulatory effects of VEGF₁₆₅ on endothelial cell proliferation, migration, and signal transduction were not altered by FSAP. Similarly, proliferation of VEGF receptor-expressing BAF3 cells, in response to VEGF₁₆₅, was not modulated by FSAP. In the mouse matrigel model of angiogenesis, FSAP decreased the ability of VEGF₁₆₅, basic fibroblast growth factor (bFGF), and their combination, to induce neovascularization. Lack of endogenous FSAP in mice did not influence neovascularization. Thus, FSAP inhibited VEGF₁₆₅-mediated angiogenesis in the matrigel model in vivo, where VEGF’s interaction with the matrix and its diffusion are important. Full article

(This article belongs to the Special Issue Arteriogenesis and Therapeutic Neovascularization)

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Review

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32 pages, 3133 KiB

Open AccessReview

Why Should Growth Hormone (GH) Be Considered a Promising Therapeutic Agent for Arteriogenesis? Insights from the GHAS Trial

by Diego Caicedo, Pablo Devesa, Clara V. Alvarez and Jesús Devesa

Cells 2020, 9(4), 807; https://doi.org/10.3390/cells9040807 - 27 Mar 2020

Cited by 13 | Viewed by 4082

Abstract

Despite the important role that the growth hormone (GH)/IGF-I axis plays in vascular homeostasis, these kind of growth factors barely appear in articles addressing the neovascularization process. Currently, the vascular endothelium is considered as an authentic gland of internal secretion due to the [...] Read more.

Despite the important role that the growth hormone (GH)/IGF-I axis plays in vascular homeostasis, these kind of growth factors barely appear in articles addressing the neovascularization process. Currently, the vascular endothelium is considered as an authentic gland of internal secretion due to the wide variety of released factors and functions with local effects, including the paracrine/autocrine production of GH or IGF-I, for which the endothelium has specific receptors. In this comprehensive review, the evidence involving these proangiogenic hormones in arteriogenesis dealing with the arterial occlusion and making of them a potential therapy is described. All the elements that trigger the local and systemic production of GH/IGF-I, as well as their possible roles both in physiological and pathological conditions are analyzed. All of the evidence is combined with important data from the GHAS trial, in which GH or a placebo were administrated to patients suffering from critical limb ischemia with no option for revascularization. We postulate that GH, alone or in combination, should be considered as a promising therapeutic agent for helping in the approach of ischemic disease. Full article

(This article belongs to the Special Issue Arteriogenesis and Therapeutic Neovascularization)

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Full article ">Figure 1
Effects of growth hormone (GH) on the vascular endothelium. The interaction GH–GHR (growth hormone receptor) produces the activation of the associated Janus kinase 2 (JAK2), which induces the phosphorylation (red circles) of tyrosine located in the cytoplasmic receptor domain, leading to the phosphorylation of GH-signaling pathways, such as signal transducers and activators of transcription (STATS). Among STATS (STATS 1 and 3 not shown in the Figure), STAT5 homodimerizes and is translocated to the nucleus, where it induces the transcription of a series of genes. Activated JAK2, acting on the insulin receptor substrate (IRS) induces the phosphorylation of phosphoinositide 3-kinase (PI3K) which, in turn, activates the cell survival factor serine-threonine kinase (Akt). This inhibits the proapoptotic enzyme Caspase 3 (red arrow), but it also activates endothelial nitric oxide synthase (eNOS) (blue arrow). Activated eNOS promotes the synthesis of nitric oxide (NO) (from L-Arginine and O2) and the formation of L-Citrulline. The formed NO flows from the cytoplasm to the muscle cell layer of the blood vessels, producing its relaxation and consequent vasodilation. The interaction GH–GHR also induces the activation of the Shc adapter proteins, which leads to the activation of the Grb2–SOS–Ras–Raf–MEK–ERK (extracellular signal-regulated kinase) pathway (Raf is not shown in the figure). Activated ERK translocates into the nucleus of the endothelial cells (ECs) and regulates the expression of genes involved in cell proliferation, differentiation, and survival, but it also regulates cell motility and migration (key for the formation of new vessels). The GH–GHR interaction also activates focal adhesion kinase (FAK). SHP: protein tyrosine phosphatase. Blue arrows: stimulation. Red arrow: inhibition. Black arrows: Translocation to the nucleus. 1: endocrine GH. 2: endothelial GH: plays and auto/paracrine role and in situations of the absence of endocrine GH perhaps plays the role of the former (black line). Full article ">Figure 2
Mitochondrial dysfunction leads to vascular endothelium senescence. An excessive production of highly reactive oxygen species (hROS) by mitochondria induces the elimination of NO, because it is transformed into peroxynitrite (ONOO–), which is toxic for endothelial cells. Moreover, the excessive oxidative stress induces the activation of nuclear factor-kappa B (NF-kB), which increases the expression of inflammatory genes in the blood vessels and decreases vasodilation. These lead to the presentation of a senescent phenotype in the endothelial cells. GH administration corrects mitochondrial dysfunction, because GH is able to enter to mitochondria and decrease the activity of complexes II and IV of the mitochondrial respiratory chain. In addition, GH produces a decrease in the mitochondrial membrane potential which translates into the release of cytochrome C (cyt C) to the cytosol. Blue arrows: stimulation. Red arrows: inhibition or damage (in endothelial cells). Orange arrow: indicates that an increase in hROS induces the transformation of NO into ONOO-. Full article ">Figure 3
GH/IGF-I are also produced in response to shear stress forces (SSF) for collateral enlargement. Schematic representation of the activation of several molecules after the increase in SSF in which GH and IGF-1 have been added as new elements that enhance these mechanisms. Since GH and IGF-1 are potent mitogenic agents, they have to play a role in the translation of mechanosensing signals. The NO pathway not only is important for vasodilation, as it also has many actions in chemoattraction of inflammatory cells and vasopermeability. The local production of cytokines and hormones seems to be essential for collateral enlargement. SSF: shear stress forces; SSRE: shear stress response elements; eNOS: endothelial nitric oxide synthase; MCP-1: monocyte chemoattractant protein-1; CAM: cellular adhesion molecules; NO: nitric oxide. Full article ">Figure 4
Signaling cascades for collateral growth: The redox system can play a very important role. Schematic representation of signaling cascades after an arterial occlusion, triggering both the inflammatory response and the phenomena of the proliferation and migration of endothelial cells into the artery media layer. Several pathways have been highlighted: Rho, involved in endothelial cells proliferation; Ras, for endothelial cells migration; and NO, for endothelial function and monocytes adhesion. The oxidative radicals produced in cellular metabolism are currently considered very important in the stimulation of the NO pathway for vascular homeostasis. They control NO bioavailability. The Rho pathway has been advocated as crucial for sensing of SSF. Caveolins: family of integral membrane proteins that play a role in the signaling of integrins and in the migration of endothelial cells. Cation channels, mainly the Ca2+ ion, are also related to the activation of Protein kinase C (PKC) and the RAS/RAC (rats’ sarcoma-extracellular signal-regulated kinases/Ras-related C3 botulinum toxin substrate). All signaling cascades are activated by SSF. For more details, see reference [<a href="#B106-cells-09-00807" class="html-bibr">106</a>,<a href="#B107-cells-09-00807" class="html-bibr">107</a>]. NADPH oxidase: nicotinamide adenine dinucleotide phosphate oxidase; NO: nitric oxide; PK-C: protein kinase C, Ras: rats’ sarcoma-extracellular signal-regulated kinases (currently known as GTPase Ras); Rho, hexameric protein found in prokaryotes, necessary for the process of terminating the transcription of some genes, Rac: Ras-related C3 botulinum toxin substrate (subfamily of the Rho family); NF-kB: nuclear factor-kappa B. MAPK: mitogen-activated protein kinase. Full article ">Figure 5
GH decreases the redox stress in ischemic muscles from the calf of patients with critical limb ischemia. Insights from the GHAS trial. (a): shows the significant decrease in NOX4 mRNA levels only seen in the GH-treated group (A, green bars) and not in Placebo-treated group (B, blue bars). (b): depicts the significant increase in the levels of NOS3 (eNOS) mRNA during the period of treatment in patients with placebo related to its baseline levels, while in the GH group (A), although there was also an increase in NOS3 mRNA levels after the treatment, this increase was not significant. Samples from soleus muscle. Group A: GH; Group B: placebo. Basal: baseline mRNA levels before treatment; final: final mRNA levels after 8 weeks of treatment. Statistics: non-parametric test. Note that of the 36 patients initially recruited for the study, complete muscle samples (basal and final) were only obtained from 28 of them as a result of deaths, limb amputations, or the patient´s refusal to allow for the second biopsy. * indicates statistically significant. Full article ">Figure 6
GH increases VEFGA-R2 or KDR/Flk-1 mRNA levels in ischemic muscles of patients with critical limb ischemia without apparent changes in IGF-I and vascular endothelial growth factor A (VEGFA) mRNA levels. Insights from the GHAS trial. (A). VEFG-R2 or KDR/Flk-1 mRNA levels in the GHAS trial. The left graph shows the baseline levels between both groups of treatment with no significant differences. In the right graph, a significant increase in KDR mRNA levels in the GH group compared to the placebo group were found during the period of treatment (8 weeks). (B). IGF-I mRNA levels in the GHAS trial. The graph on the left shows levels of IGF-I at baseline in ischemic vs non-ischemic muscle samples with no differences. The graph on the right shows the lack of changes in any group of treatment during the period of treatment. (C). VEGFA mRNA levels in the GHAS trial. The left graph depicts the comparation between VEGFA levels in ischemic vs non-ischemic muscle samples in the calf at baseline. The right graph depicts the lack of significant changes after 8 weeks of treatment. Group A, green bars: GH; Group B, blue bars: placebo. Non-ischemic muscle samples: sample of reference obtained from amputations in patients without limb ischemia. Commercial pool mRNA: a commercial mRNA from skeletal muscle used as a technical control for normalization of the different assays. A non-parametric test was used for the statistics. Note that although at the beginning of the study there were data from 35 muscle samples, at the end of this study, there were only complete data from 28 patients (basal and final samples). * indicates statistically significant. Full article ">

15 pages, 567 KiB

Open AccessReview

Arteriogenesis of the Spinal Cord—The Network Challenge

by Florian Simon, Markus Udo Wagenhäuser, Albert Busch, Hubert Schelzig and Alexander Gombert

Cells 2020, 9(2), 501; https://doi.org/10.3390/cells9020501 - 22 Feb 2020

Cited by 18 | Viewed by 6888

Abstract

Spinal cord ischemia (SCI) is a clinical complication following aortic repair that significantly impairs the quality and expectancy of life. Despite some strategies, like cerebrospinal fluid drainage, the occurrence of neurological symptoms, such as paraplegia and paraparesis, remains unpredictable. Beside the major blood [...] Read more.

Spinal cord ischemia (SCI) is a clinical complication following aortic repair that significantly impairs the quality and expectancy of life. Despite some strategies, like cerebrospinal fluid drainage, the occurrence of neurological symptoms, such as paraplegia and paraparesis, remains unpredictable. Beside the major blood supply through conduit arteries, a huge collateral network protects the central nervous system from ischemia—the paraspinous and the intraspinal compartment. The intraspinal arcades maintain perfusion pressure following a sudden inflow interruption, whereas the paraspinal system first needs to undergo arteriogenesis to ensure sufficient blood supply after an acute ischemic insult. The so-called steal phenomenon can even worsen the postoperative situation by causing the hypoperfusion of the spine when, shortly after thoracoabdominal aortic aneurysm (TAAA) surgery, muscles connected with the network divert blood and cause additional stress. Vessels are a conglomeration of different cell types involved in adapting to stress, like endothelial cells, smooth muscle cells, and pericytes. This adaption to stress is subdivided in three phases—initiation, growth, and the maturation phase. In fields of endovascular aortic aneurysm repair, pre-operative selective segmental artery occlusion may enable the development of a sufficient collateral network by stimulating collateral vessel growth, which, again, may prevent spinal cord ischemia. Among others, the major signaling pathways include the phosphoinositide 3 kinase (PI3K) pathway/the antiapoptotic kinase (AKT) pathway/the endothelial nitric oxide synthase (eNOS) pathway, the Erk1, the delta-like ligand (DII), the jagged (Jag)/NOTCH pathway, and the midkine regulatory cytokine signaling pathways. Full article

(This article belongs to the Special Issue Arteriogenesis and Therapeutic Neovascularization)

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12 pages, 744 KiB

Open AccessReview

Exercise-Induced Vascular Adaptations under Artificially Versus Pathologically Reduced Blood Flow: A Focus Review with Special Emphasis on Arteriogenesis

by Johanna Vogel, Daniel Niederer, Georg Jung and Kerstin Troidl

Cells 2020, 9(2), 333; https://doi.org/10.3390/cells9020333 - 31 Jan 2020

Cited by 10 | Viewed by 3905

Abstract

Background: The vascular effects of training under blood flow restriction (BFR) in healthy persons can serve as a model for the exercise mechanism in lower extremity arterial disease (LEAD) patients. Both mechanisms are, inter alia, characterized by lower blood flow in the lower [...] Read more.

Background: The vascular effects of training under blood flow restriction (BFR) in healthy persons can serve as a model for the exercise mechanism in lower extremity arterial disease (LEAD) patients. Both mechanisms are, inter alia, characterized by lower blood flow in the lower limbs. We aimed to describe and compare the underlying mechanism of exercise-induced effects of disease- and external application-BFR methods. Methods: We completed a narrative focus review after systematic literature research. We included only studies on healthy participants or those with LEAD. Both male and female adults were considered eligible. The target intervention was exercise with a reduced blood flow due to disease or external application. Results: We identified 416 publications. After the application of inclusion and exclusion criteria, 39 manuscripts were included in the vascular adaption part. Major mechanisms involving exercise-mediated benefits in treating LEAD included: inflammatory processes suppression, proinflammatory immune cells, improvement of endothelial function, remodeling of skeletal muscle, and additional vascularization (arteriogenesis). Mechanisms resulting from external BFR application included: increased release of anabolic growth factors, stimulated muscle protein synthesis, higher concentrations of heat shock proteins and nitric oxide synthase, lower levels in myostatin, and stimulation of S6K1. Conclusions: A main difference between the two comparators is the venous blood return, which is restricted in BFR but not in LEAD. Major similarities include the overall ischemic situation, the changes in microRNA (miRNA) expression, and the increased production of NOS with their associated arteriogenesis after training with BFR. Full article

(This article belongs to the Special Issue Arteriogenesis and Therapeutic Neovascularization)

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22 pages, 1257 KiB

Open AccessReview

An Emerging Role for isomiRs and the microRNA Epitranscriptome in Neovascularization

by Reginald V.C.T. van der Kwast, Paul H.A. Quax and A. Yaël Nossent

Cells 2020, 9(1), 61; https://doi.org/10.3390/cells9010061 - 25 Dec 2019

Cited by 28 | Viewed by 5417

Abstract

Therapeutic neovascularization can facilitate blood flow recovery in patients with ischemic cardiovascular disease, the leading cause of death worldwide. Neovascularization encompasses both angiogenesis, the sprouting of new capillaries from existing vessels, and arteriogenesis, the maturation of preexisting collateral arterioles into fully functional arteries. [...] Read more.

Therapeutic neovascularization can facilitate blood flow recovery in patients with ischemic cardiovascular disease, the leading cause of death worldwide. Neovascularization encompasses both angiogenesis, the sprouting of new capillaries from existing vessels, and arteriogenesis, the maturation of preexisting collateral arterioles into fully functional arteries. Both angiogenesis and arteriogenesis are highly multifactorial processes that require a multifactorial regulator to be stimulated simultaneously. MicroRNAs can regulate both angiogenesis and arteriogenesis due to their ability to modulate expression of many genes simultaneously. Recent studies have revealed that many microRNAs have variants with altered terminal sequences, known as isomiRs. Additionally, endogenous microRNAs have been identified that carry biochemically modified nucleotides, revealing a dynamic microRNA epitranscriptome. Both types of microRNA alterations were shown to be dynamically regulated in response to ischemia and are able to influence neovascularization by affecting the microRNA’s biogenesis, or even its silencing activity. Therefore, these novel regulatory layers influence microRNA functioning and could provide new opportunities to stimulate neovascularization. In this review we will highlight the formation and function of isomiRs and various forms of microRNA modifications, and discuss recent findings that demonstrate that both isomiRs and microRNA modifications directly affect neovascularization and vascular remodeling. Full article

(This article belongs to the Special Issue Arteriogenesis and Therapeutic Neovascularization)

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13 pages, 683 KiB

Open AccessReview

Atherosclerosis and the Capillary Network; Pathophysiology and Potential Therapeutic Strategies

by Tilman Ziegler, Farah Abdel Rahman, Victoria Jurisch and Christian Kupatt

Cells 2020, 9(1), 50; https://doi.org/10.3390/cells9010050 - 24 Dec 2019

Cited by 51 | Viewed by 8408

Abstract

Atherosclerosis and associated ischemic organ dysfunction represent the number one cause of mortality worldwide. While the key drivers of atherosclerosis, arterial hypertension, hypercholesterolemia and diabetes mellitus, are well known disease entities and their contribution to the formation of atherosclerotic plaques are intensively studied [...] Read more.

Atherosclerosis and associated ischemic organ dysfunction represent the number one cause of mortality worldwide. While the key drivers of atherosclerosis, arterial hypertension, hypercholesterolemia and diabetes mellitus, are well known disease entities and their contribution to the formation of atherosclerotic plaques are intensively studied and well understood, less effort is put on the effect of these disease states on microvascular structure an integrity. In this review we summarize the pathological changes occurring in the vascular system in response to prolonged exposure to these major risk factors, with a particular focus on the differences between these pathological alterations of the vessel wall in larger arteries as compared to the microcirculation. Furthermore, we intend to highlight potential therapeutic strategies to improve microvascular function during atherosclerotic vessel disease. Full article

(This article belongs to the Special Issue Arteriogenesis and Therapeutic Neovascularization)

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Figure 1
(A) The healthy circulatory system is characterized by minimal lipid accumulation in larger arteries and an overall low state of endothelial activation, leading to low levels of ROS production and leukocyte recruitment. (B) Upon prolonged exposure to the atherosclerotic risk factors arterial hypertension, hypercholesterolemia and diabetes, endothelial cells experience constant activation enhancing leukocyte recruitment, oxidative stress and loss of pericytes in the microcirculation, leading to capillary rarefication, limiting the potential blood flow through the now sparse capillary network. (C) Even after mechanical revascularization, via bypass operations or percutaneous angioplasty, the capillary rarefication remains, continuously limiting blood flow, thus hindering the recovery of the ischemic tissue and leaving newly opened vessels susceptible for restenosis and stent thrombosis. Here, strategies to improve capillary density, and thus, microcirculatory flow, appear to be worthwhile therapeutic targets in the treatment of atherosclerosis currently not yet addresses. Full article ">Figure 2
Effect of in vivo genome editing via rAAV and Cas9 mediated deletion of exon 51 of Duchenne muscular dystrophy on the vascularization and macrophage recruitment in the heart and upper and lower hind limb in DMDΔ52 pigs. (A) staining for CD31 positive endothelial cells highlights a significant decrease in capillary density in pigs suffering from Duchenne muscular dystrophy which ins ameliorated in pigs receiving Cas9 mediated Exon 51 deletion thus restoring dystrophin expression. (B) Similarly, edited DMD pigs display an amelioration of pericyte loss seen in dystrophin deficient pigs. (C) Lastly, the reduction in both endothelial cells as well as pericytes in dystrophin deficient pigs is accompanied by an increased recruitment of CD68 positive macrophages into the tissue, similarly to the recruitment seen in atherosclerotic states, which is again reversed upon normalization of dystrophin expression (*p < 0.05 versus wild type and DMD+rAAV.Cas9, error bars are given as SEM). Full article ">

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