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. 2020 Dec 21;8(4):2002500.
doi: 10.1002/advs.202002500. eCollection 2021 Feb.

Nidogen-1 Mitigates Ischemia and Promotes Tissue Survival and Regeneration

Aline Zbinden  1   2 , Shannon L Layland  1   2 , Max Urbanczyk  1   2 , Daniel A Carvajal Berrio  1   2   3 , Julia Marzi  1   2   3   4 , Monika Zauner  2 , Anne Hammerschmidt  2 , Eva M Brauchle  2   3   4 , Katrin Sudrow  5   6 , Simon Fink  4 , Markus Templin  4 , Simone Liebscher  1   2 , Gerd Klein  7 , Arjun Deb  8   9   10   11   12 , Garry P Duffy  13 , Gay M Crooks  9   11   14 , Johannes A Eble  15 , Hanna K A Mikkola  8   9   10   11 , Ali Nsair  9   12 , Martina Seifert  5   6   16 , Katja Schenke-Layland  1   2   3   4   12
Affiliations

Nidogen-1 Mitigates Ischemia and Promotes Tissue Survival and Regeneration

Aline Zbinden et al. Adv Sci (Weinh). .

Abstract

Ischemia impacts multiple organ systems and is the major cause of morbidity and mortality in the developed world. Ischemia disrupts tissue homeostasis, driving cell death, and damages tissue structure integrity. Strategies to heal organs, like the infarcted heart, or to replace cells, as done in pancreatic islet β-cell transplantations, are often hindered by ischemic conditions. Here, it is discovered that the basement membrane glycoprotein nidogen-1 attenuates the apoptotic effect of hypoxia in cardiomyocytes and pancreatic β-cells via the αvβ3 integrin and beneficially modulates immune responses in vitro. It is shown that nidogen-1 significantly increases heart function and angiogenesis, while reducing fibrosis, in a mouse postmyocardial infarction model. These results demonstrate the protective and regenerative potential of nidogen-1 in ischemic conditions.

Keywords: diabetes; ischemia; myocardial infarction; nidogen‐1; pancreatic β‐cells.

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Conflict of interest statement

A.Z., S.L.L., G.P.D., and K.S.‐L. are inventors on patent application EP19154849.4 associated with this work and owned by the University Tübingen. S.L.L., M.Z., and K.S.‐L. are inventors on patents (EP3027201B1 and CN105517564A), and patent applications (US20160158314A1, CA2916614A1, JP2016530532A, and KR20160037170A) associated with this work and owned by the NMI, Reutlingen.

Figures

Figure 1
Figure 1
ECM BM protein NID1 is identified as a candidate for regenerative approaches. A) IF staining and semiquantification of BM proteins NID1, FN, POSTN, LAM, COL4, and COL1, as well as DAPI and MF20 in day‐10 beating EBs. Gray value intensities (GVI) of IF images were normalized to the laser intensity (n = 3), one‐way ANOVA with Tukey's multiple comparisons test. B) qPCR analysis of NID1 expression on day 0 (undifferentiated hESCs), and after 4 and 10 days (beating EBs) of cardiovascular differentiation. Data are normalized to the average of day 0 and shown as fold change (n = 3–4); one‐way ANOVA with Tukey's multiple comparisons test. C) qPCR analysis of TNNT2 and ACTA2 gene expression within EBs at day 10 of cardiovascular differentiation without (control) and with NID1 (n = 5); Kolmogorov–Smirnov t‐test. D) Bright field (BF) and CTNT IF images of single cells derived from EBs that were cultured for 10 days without (control) and with NID1 acquired with 40× magnification using imaging flow cytometry. An isotype control is provided. E) Representative histogram of cells that were cultured for 10 days with (blue) or without (red) NID1. The isotype control is shown in gray. F) Quantification of the data obtained by imaging flow cytometric analysis showing the relative amount of CTNT+ cells derived from EBs that were cultured for 10 days without (control) and with NID1 (n = 7); Kolmogorov–Smirnov t‐test. G,H) IF staining of BM proteins COL4, LAM, NID1, NID2, as well as DAPI and sarcomeric myosin CTNT within human (G) fetal heart sections (9 weeks postgestation) and (H) adult heart tissue (51 years). Scale bars: 20 µm. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 2
Figure 2
NID1 increases heart function post‐MI. A–C) Echocardiography analysis: absolute values of (A) EF and (B) FS after intracardiac injections of saline, HA and 50 µg mL−1 NID1 + HA at 28 days post‐MI/R, and C) parameters were normalized to the baseline at 28 days post‐MI/R. Echocardiography data were analyzed by one‐way ANOVA with Tukey's multiple comparisons test. D–I) Movat pentachrome staining of representative sections of (D–F) HA‐ and (G–I) NID1 + HA‐treated hearts after 28 days post‐MI/R with scar tissue stained in green and yellow. Scale bars: (D,G) 1 mm, (E,H) 200 µm, and (F,I) 100 µm. J,K) Picrosirius Red and Fast Green staining of representative (J) HA‐ and (K) NID1 + HA‐treated heart sections with scar tissue stained in pink. Scale bars: 1 mm. L) Quantification of scar size in Picrosirius Red‐ and Fast Green‐stained serial sections. Whole‐heart scans of every tenth slide throughout the whole heart were analyzed. M–P) Confocal images of αSMA, CD31, and CTNT IF staining of representative (M) baseline, (N) HA‐, and (O) NID1 + HA‐treated heart sections obtained with a 25× magnification (scale bar: 100 µm), and with a P) 63× magnification (scale bar; 50 µm). Q) Quantification of vessel density within the infarct area using images obtained with a 25× magnification. R–U) Images of TuJ1 and CTNT IF staining of representative (R) baseline, (S) HA‐, and (T) NID1 + HA‐treated tissue sections obtained with a 25× magnification (scale bar equal 100 µm), and a (U) 63× magnification (scale bar: 50 µm). V) Quantification of TuJ1+ cells within the infarct area. For all MI/R studies saline mice (n = 3), HA mice (n = 3), NID1 + HA‐treated mice (n = 4) were used, unpaired t‐test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. LVIDd: left ventricular internal dimension at end diastole, LVIDs: left ventricular internal dimension at end‐systole, LVED: left ventricle end‐diastolic diameter, LVES: left ventricle end‐systolic diameter, EDV: end‐diastolic volume, ESV: end‐systolic volume, EF: ejection fraction, FS: fractional shortening.
Figure 3
Figure 3
NID1 mitigates the effects of hypoxia on cardiovascular cells. A,B) Protective effect of NID1 on hiPSC‐CMs shown by (A) cleaved caspase‐3 staining and semiquantification (n = 6) and (B) by number of TUNEL+ cells over total cell number (n = 10); one‐way ANOVA with Tukey's multiple comparisons test. C) qPCR array analysis of NID1‐treated hiPSC‐CMs under hypoxic conditions with a focus on matrix production, degradation, and regulation (n = 3). D) qPCR analysis for the expression of αSMA in control fibroblasts under normoxic and hypoxic conditions, as well as for NID1‐treated fibroblasts under hypoxic conditions (n = 3). One‐way ANOVA with Tukey's multiple comparisons test. E) qPCR array analysis of NID1‐treated fibroblasts under hypoxic conditions (identical qPCR array as in (C)) (n = 3). F) Tube formation assay performed using 1.5 × 104 HUVECs/0.32 cm2 with serum‐reduced Matrigel without (control) or with NID1 (n = 4–5). Quantification of different tube formation parameters using the angiogenesis analyzer of the ImageJ software. Data are normalized to the Matrigel control (set as 1), one‐way ANOVA with Tukey's multiple comparisons test. For qPCR, gene regulation by NID1 is shown when fold‐regulation > |2|, p‐value < 0.05. *p < 0.05; **p < 0.01, ***p < 0.001, and ****p < 0.0001. Scale bars: 50 µm.
Figure 4
Figure 4
NID1 mitigates the effects of hypoxia on pancreatic β‐cells and increases insulin secretion. A,B) Expression pattern of BM proteins, insulin (INS), and DAPI in human (A) fetal (11 weeks postgestation) and (B) adult pancreas (64 years). Scale bars: 20 µm. Highly magnified images show the colocalization of NID1 with INS in native adult pancreas. Scale bar: 5 µm. C) Quantification of the colocalization of ECM proteins with INS (n = 10), one‐way ANOVA with Tukey's multiple comparisons test. D) GSIS response (with 0 × 10−3 and 20 × 10−3 m glucose) under normoxic conditions of human NID1‐treated pseudoislets in suspension at different concentrations: 20, 30, 40 µg mL−1 when compared with the control (PBS) (n = 5); two‐way ANOVA with Tukey's multiple comparisons test. E) E‐cadherin expression under normoxic conditions (n ≥ 5). F) Cell death under normoxic conditions via the detection of cleaved caspase‐3 (n = 14) and TUNEL+ cells (n = 7). G) GSIS response (with 0 × 10−3 and 20 × 10−3 m glucose) under hypoxic conditions of NID1‐treated pseudoislets at 30 µg mL−1 and normalized by live cells (n = 10); two‐way ANOVA with Tukey's multiple comparisons test. H) E‐cadherin expression under hypoxic conditions (n ≥ 5 m; unpaired t‐test. I) Protective effect of NID1 assessed by cleaved caspase‐3 expression (n ≥ 7). and via detection of TUNEL+ cells (n ≥ 4); unpaired t‐test. *p < 0.05; **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 5
Figure 5
NID1 modulates immune cells. A) CD14+ monocyte migration in the presence of different NID1 concentrations (50, 100 µg mL−1) and the control protein MCP‐1 after 3 h. Cell migration is shown relative to the control (−) (n = 3 experiments with 9 single values); one‐way ANOVA with Dunnett's multiple comparison test. B) Potential endotoxin contamination was tested by adding 50 µg mL−1 NID1 or 100 ng mL−1 LPS in a monocyte culture for 24 h. Released TNFα was measured by ELISA (n = 5); Friedman test with Dunn's multiple comparison test. C) Representative images of M1‐ and M2‐type macrophages, unstimulated M0‐type macrophages and NID1‐treated M0‐type macrophages. Scale bar: 100 µm. D) Expression of polarization markers (HLA‐DR, CD80, CD206, CD163) was determined for M0‐type macrophages cultured for 24 h with 50 µg mL−1 NID1, M0, M1, and M2‐type macrophages. Mean fluorescence intensity (MFI) is shown relative to the untreated M0 macrophage control (set as 1) (n = 5); two‐way ANOVA with Dunnett's multiple comparison test. E) Release of IL‐6, TNFα, and IL‐10 after 24 h culture of M0 macrophages with NID1 (50 µg mL−1) and M0, M1 or M2 type cultures (n = 5); two‐way ANOVA with Tukey's multiple comparison post‐test. F) T cell proliferation after 5 days culture of human PBMCs with low‐dose aCD3 alone or combined with NID1 (50 µg mL−1) was tested in a CFSE‐based assay and measured by flow cytometry. Shown is the relative proliferation level of CD3+ CD4+ and CD3+ CD8+ T cells compared to the aCD3 control. G) Relative TNFα and IFNγ release of PBMC cultures after 5 days with either aCD3 alone or combined with NID1 (50 µg mL−1). Data for proliferation and cytokine release were analyzed by Kolmogorov–Smirnov t‐test, relative to the aCD3 control (n = 6). H) Cytotoxicity test using human dermal fibroblasts (EN ISO 10993) or I) cardiac fibroblasts. Cell viability was measured after treatment with control medium, HA hydrogel, and HA‐supplemented with NID1 (50, 100, and 200 µg mL−1) for 24 h. Cell viability is shown relative to the control (set as 100% viability) (n ≥ 22); one‐way ANOVA with Tukey's multiple comparisons test: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Figure 6
Figure 6
NID1 signals via αvβ3 and activates the MAPK pathway in β‐cells and cardiomyocytes. A) Divalent cation‐dependent and dose‐dependent binding of αvβ3 with NID1. B) Blocking of αvβ3 in NID1‐treated and control pseudoislets under normoxic conditions. Functionality assessment by glucose stimulation at 20 × 10−3 m glucose (n ≥ 4); one‐way ANOVA with Tukey's multiple comparisons test. c,d) Blocking of αvβ3 in NID1‐treated and control hiPSC‐CMs by a αvβ3 antibody under hypoxic conditions. Protective effect of NID1 assessed by C) cleaved caspase‐3 (n ≥ 4) and D) TUNEL assay (n = 6); one‐way ANOVA with Tukey's multiple comparisons test. E–H) DigiWest‐based protein expression analysis of significantly regulated proteins in NID1‐treated E,F) pseudoislets (n = 12) and G,H) hiPSC‐CMs (n = 4) under hypoxic conditions compared with their respective controls. Data are (E,G) shown as column‐wise and color‐coded heatmap from the lowest (blue) to the highest (yellow) expression for each analyte; and (F,H) quantified as fold change induced by NID1. Nonparametric Wilcoxon Rank sum test. I,J) Protein ratios of (I) Bax to Bcl2 and (J) SAPK to Erk1,2 (n = 4); unpaired t‐test. K) Proposed common and separate mechanisms of action of NID1 for CMs and β‐cells in vitro. Middle: common pathway, NID1‐αvβ3 ligation upregulating Fyn/Src in β‐cells and cdc42 in CMs, which stimulates Wnt3 and the MAPK pathway. Left: β‐cells, Fyn/Src activates the MAPK pathway, upregulating p21, which can be antiapoptotic. Fyn/Src crosstalks with Wnt3 and EpCAM, which enhances insulin secretion. Right: CMs, cdc42 downregulates the Hippo pathway as shown by an upregulation of MOB1. Pathways specific for each cell type are shown either on the left for β‐cells and on the right for CMs. *p < 0.05; **p < 0.01, ***p < 0.001, and ****p < 0.0001.
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