Publications by authors named "Rob C M de Jong"

12 Publications

  • Page 1 of 1

Phosphorylcholine antibodies restrict infarct size and left ventricular remodelling by attenuating the unreperfused post-ischaemic inflammatory response.

J Cell Mol Med 2021 Aug 30;25(16):7772-7782. Epub 2021 Jun 30.

Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands.

Phosphorylcholine is a pro-inflammatory epitope exposed on apoptotic cells, and phosphorylcholine monoclonal immunoglobulin (Ig)G antibodies (PC-mAb) have anti-inflammatory properties. In this study, we hypothesize that PC-mAb treatment reduces adverse cardiac remodelling and infarct size (IS) following unreperfused transmural myocardial infarction (MI). Unreperfused MI was induced by permanent ligation of the left anterior descending (LAD) coronary artery in hypercholesterolaemic APOE*3-Leiden mice. Three weeks following MI, cardiac magnetic resonance (CMR) imaging showed a reduced LV end-diastolic volume (EDV) by 21% and IS by 31% upon PC-mAb treatment as compared to the vehicle control group. In addition, the LV fibrous content was decreased by 27% and LV wall thickness was better preserved by 47% as determined by histological analysis. Two days following MI, CCL2 concentrations, assessed by use of ELISA, were decreased by 81% and circulating monocytes by 64% as assessed by use of FACS analysis. Additionally, local leucocyte infiltration determined by immunohistological analysis showed a 62% decrease after three weeks. In conclusion, the local and systemic inflammatory responses are limited by PC-mAb treatment resulting in restricted adverse cardiac remodelling and IS following unreperfused MI. This indicates that PC-mAb holds promise as a therapeutic agent following MI limiting adverse cardiac remodelling.
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http://dx.doi.org/10.1111/jcmm.16662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8358891PMC
August 2021

P300/CBP Associated Factor (PCAF) Deficiency Enhances Diet-Induced Atherosclerosis in ApoE3Leiden Mice via Systemic Inhibition of Regulatory T Cells.

Front Cardiovasc Med 2020 15;7:604821. Epub 2021 Jan 15.

Department of Surgery, Leiden University Medical Center, Leiden, Netherlands.

Inflammatory stimuli induced by NF-kB drive atherosclerotic lesion formation. The epigenetic P300/CBP associated factor (PCAF) post-transcriptionally acetylates FoxP3, which is required for regulatory T-cell (Treg) differentiation and immune modulation. We hypothesize that PCAF deficiency affects atherosclerosis via regulation of regulatory Tregs. ApoE3Leiden ( = 13) and ApoE3LeidenxPCAF ( = 13) were fed a high-fat diet (HFD) containing 1.25% cholesterol. Systemic FoxP3 T cells were measured every 4 weeks by flow cytometry ( = 6). After 5-months of HFD, mice were euthanized, and hearts and blood were collected. IL-6 and TNFα concentrations were measured in plasma to identify systemic inflammatory responses. Compositional and morphometrical analyses were performed on the atherosclerotic lesions in the aortic sinuses. After 5 months of HFD, plasma cholesterol concentrations were not different for ApoE3LeidenxPCAF compared to ApoE3Leiden mice. Expression of FoxP3 by systemic CD4 T cells decreased 1.8 fold in ApoE3LeidenxPCAF after 5 months HFD and remained significantly reduced after 5 months of HFD. Systemic TNFα and IL-6 concentrations were comparable, whereas the atherosclerotic lesion size in ApoE3LeidenxPCAF mice was increased by 28% compared to ApoE3Leiden mice. In atherosclerotic lesions, no differences were observed in macrophage differentiation or VSMC content, although a small increase in collagen was identified. Our data show that PCAF deficiency resulted in a decrease in circulatory FoxP3 regulatory T cells and ameliorated atherosclerotic lesions with no differences in systemic inflammation or macrophage differentiation in the atherosclerotic lesions. This suggests that PCAF regulates atherosclerosis via modulation of FoxP3 regulatory T cell differentiation.
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http://dx.doi.org/10.3389/fcvm.2020.604821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7874080PMC
January 2021

Atorvastatin pleiotropically decreases intraplaque angiogenesis and intraplaque haemorrhage by inhibiting ANGPT2 release and VE-Cadherin internalization.

Angiogenesis 2021 08 7;24(3):567-581. Epub 2021 Feb 7.

Department of Vascular Surgery, Leiden University Medical Center, Leiden, The Netherlands.

Objective: Statins pleiotropically provide additional benefits in reducing atherosclerosis, but their effects on intraplaque angiogenesis (IPA) and hemorrhage (IPH) remain unclear. Therefore, we discriminated statin's lipid-lowering dependent and independent effects on IPA and IPH.

Approach And Results: ApoE3*Leiden mice are statin-responsive due to ApoE and LDLR presence, but also allow to titrate plasma cholesterol levels by diet. Therefore, ApoE3*Leiden mice were fed a high-cholesterol-inducing-diet (HCD) with or without atorvastatin (A) or a moderate-cholesterol-inducing-diet (MCD). Mice underwent vein graft surgery to induce lesions with IPA and IPH. Cholesterol levels were significantly reduced in MCD (56%) and HCD + A (39%) compared to HCD with no significant differences between MCD and HCD + A. Both MCD and HCD + A have a similar reduction in vessel remodeling and inflammation comparing to HCD. IPA was significantly decreased by 30% in HCD + A compared to HCD or MCD. Atorvastatin treatment reduced the presence of immature vessels by 34% vs. HCD and by 25% vs. MCD, resulting in a significant reduction of IPH. Atorvastatin's anti-angiogenic capacity was further illustrated by a dose-dependent reduction of ECs proliferation and migration. Cultured mouse aortic-segments lost sprouting capacity upon atorvastatin treatment and became 30% richer in VE-Cadherin expression and pericyte coverage. Moreover, Atorvastatin inhibited ANGPT2 release and decreased VE-Cadherin(Y685)-phosphorylation in ECs.

Conclusions: Atorvastatin has beneficial effects on vessel remodeling due to its lipid-lowering capacity. Atorvastatin has strong pleiotropic effects on IPA by decreasing the number of neovessels and on IPH by increasing vessel maturation. Atorvastatin improves vessel maturation by inhibiting ANGPT2 release and phospho(Y658)-mediated VE-Cadherin internalization.
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http://dx.doi.org/10.1007/s10456-021-09767-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8292290PMC
August 2021

Phosphorylcholine Antibodies Preserve Cardiac Function and Reduce Infarct Size by Attenuating the Post-Ischemic Inflammatory Response.

JACC Basic Transl Sci 2020 Dec 2;5(12):1228-1239. Epub 2020 Dec 2.

Department of Surgery, Leiden University Medical Center, Leiden, the Netherlands.

Phosphorylcholine monoclonal immunoglobulin G antibody attenuates the immediate post-ischemic inflammatory response by reducing the proinflammatory chemokine (C-C motif) ligand 2 chemokine and circulating Ly-6C monocytes. This subsequently enhances the post-ischemic repair process, resulting in limited adverse cardiac remodeling and preservation of cardiac function. Therefore, phosphorylcholine monoclonal immunoglobulin G antibody therapy may be a valid therapeutic approach against myocardial ischemia-reperfusion injury.
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http://dx.doi.org/10.1016/j.jacbts.2020.09.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7775955PMC
December 2020

Identification of Functional HLA-A*01 :01-Restricted EBV-LMP2-Specific T-cell Receptors.

J Infect Dis 2020 Aug 18. Epub 2020 Aug 18.

Department of Hematology, Leiden University Medical Center, The Netherlands.

Background: Adoptive transfer of genetically engineered T cells expressing antigen-specific T-cell receptors (TCRs), is an appealing therapeutic approach for Epstein-Barr virus (EBV)-associated malignancies of latency type II/III that express EBV-antigens (LMP1/2). Patients who are HLA-A*01:01pos could benefit from such products, since no T cells recognizing any EBV-derived peptide in this common HLA allele have been found thus far.

Methods: HLA-A*01:01-restricted EBV-(LMP2)-specific T-cells were isolated using peptide-MHC-tetramers. Functionality was assessed by production of IFNγ and cytotoxicity when stimulated with EBV-LMP2-expressing cell-lines. Functionality of primary T cells transduced with HLA-A*01:01-restricted EBV-LMP2-specific TCRs was optimized by knocking out the endogenous TCR of primary T cells (ΔTCR) using CRISPR-Cas9 technology.

Results: EBV-LMP2-specific T cells were successfully isolated and their TCRs were characterized. TCR gene-transfer in primary T cells resulted in specific peptide-MHC-tetramer binding and reactivity against EBV-LMP2-expressing cell-lines. The mean-fluorescence intensity of peptide-MHC-tetramer binding was increased 1.5-2 fold when the endogenous TCR of CD8pos T cells was knocked out. CD8pos/ΔTCR T cells modified to express EBV-LMP2-specific TCRs showed IFNγ secretion and cytotoxicity towards EBV-LMP2-expressing malignant cell-lines.

Discussion: We isolated the first functional HLA-A*01:01-restricted EBV-LMP2-specific T-cell populations and TCRs, which can potentially be used in future TCR gene-therapy to treat EBV-associated latency type II/III malignancies.
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http://dx.doi.org/10.1093/infdis/jiaa512DOI Listing
August 2020

IRF3 and IRF7 mediate neovascularization via inflammatory cytokines.

J Cell Mol Med 2019 06 1;23(6):3888-3896. Epub 2019 Apr 1.

Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands.

Objective: To elucidate the role of interferon regulatory factor (IRF)3 and IRF7 in neovascularization.

Methods: Unilateral hind limb ischaemia was induced in Irf3 , Irf7 and C57BL/6 mice by ligation of the left common femoral artery. Post-ischaemic blood flow recovery in the paw was measured with laser Doppler perfusion imaging. Soleus, adductor and gastrocnemius muscles were harvested to investigate angiogenesis and arteriogenesis and inflammation.

Results: Post-ischaemic blood flow recovery was decreased in Irf3 and Irf7 mice compared to C57BL/6 mice at all time points up to and including sacrifice, 28 days after surgery (t28). This was supported by a decrease in angiogenesis and arteriogenesis in soleus and adductor muscles of Irf3 and Irf7 mice at t28. Furthermore, the number of macrophages around arterioles in adductor muscles was decreased in Irf3 and Irf7 mice at t28. In addition, mRNA expression levels of pro-inflammatory cytokines (tnfα, il6, ccl2) and growth factor receptor (vegfr2), were decreased in gastrocnemius muscles of Irf3 and Irf7 mice compared to C57BL/6 mice.

Conclusion: Deficiency of IRF3 and IRF7 results in impaired post-ischaemic blood flow recovery caused by attenuated angiogenesis and arteriogenesis linked to a lack of inflammatory components in ischaemic tissue. Therefore, IRF3 and IRF7 are essential regulators of neovascularization.
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http://dx.doi.org/10.1111/jcmm.14247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533520PMC
June 2019

Annexin A5 reduces infarct size and improves cardiac function after myocardial ischemia-reperfusion injury by suppression of the cardiac inflammatory response.

Sci Rep 2018 04 30;8(1):6753. Epub 2018 Apr 30.

Department of Surgery, Leiden University Medical Center, Albinusdreef 2, P.O. Box 9600, 2300 RC, Leiden, The Netherlands.

Annexin A5 (AnxA5) is known to have anti-inflammatory and anti-apoptotic properties. Inflammation and apoptosis are key processes in post-ischemic cardiac remodeling. In this study, we investigated the effect of AnxA5 on left ventricular (LV) function and remodeling three weeks after myocardial ischemia-reperfusion (MI-R) injury in hypercholesterolemic ApoE*3-Leiden mice. Using a mouse model for MI-R injury, we demonstrate AnxA5 treatment resulted in a 27% reduction of contrast-enhanced MRI assessed infarct size (IS). End-diastolic and end-systolic volumes were decreased by 22% and 38%, respectively. LV ejection fraction was increased by 29% in the AnxA5 group compared to vehicle. Following AnxA5 treatment LV fibrous content after three weeks was reduced by 42%, which was accompanied by an increase in LV wall thickness of the infarcted area by 17%. Two days and three weeks after MI-R injury the number of cardiac macrophages was significantly reduced in both the infarct area and border zones following AnxA5 treatment compared to vehicle treatment. Finally, we found that AnxA5 stimulation leads to a reduction of IL-6 production in bone-marrow derived macrophages in vitro. AnxA5 treatment attenuates the post-ischemic inflammatory response and ameliorates LV remodeling which improves cardiac function three weeks after MI-R injury in hypercholesterolemic ApoE*3-Leiden mice.
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http://dx.doi.org/10.1038/s41598-018-25143-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5928225PMC
April 2018

The Prebiotic Inulin Aggravates Accelerated Atherosclerosis in Hypercholesterolemic APOE*3-Leiden Mice.

Nutrients 2018 Feb 3;10(2). Epub 2018 Feb 3.

Department of Human Genetics and Einthoven Laboratory for Experimental Medicine, Leiden University Medical Center (LUMC), 2300 RC Leiden, The Netherlands.

The prebiotic inulin has proven effective at lowering inflammation and plasma lipid levels. As atherosclerosis is provoked by both inflammation and hyperlipidemia, we aimed to determine the effect of inulin supplementation on atherosclerosis development in hypercholesterolemic () mice. Male mice were fed a high-cholesterol (1%) diet, supplemented with or without 10% inulin for 5 weeks. At week 3, a non-constrictive cuff was placed around the right femoral artery to induce accelerated atherosclerosis. At week 5, vascular pathology was determined by lesion thickness, vascular remodeling, and lesion composition. Throughout the study, plasma lipids were measured and in week 5, blood monocyte subtypes were determined using flow cytometry analysis. In contrast to our hypothesis, inulin exacerbated atherosclerosis development, characterized by increased lesion formation and outward vascular remodeling. The lesions showed increased number of macrophages, smooth muscle cells, and collagen content. No effects on blood monocyte composition were found. Inulin significantly increased plasma total cholesterol levels and total cholesterol exposure. In conclusion, inulin aggravated accelerated atherosclerosis development in hypercholesterolemic mice, accompanied by adverse lesion composition and outward remodeling. This process was not accompanied by differences in blood monocyte composition, suggesting that the aggravated atherosclerosis development was driven by increased plasma cholesterol.
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http://dx.doi.org/10.3390/nu10020172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5852748PMC
February 2018

The epigenetic factor PCAF regulates vascular inflammation and is essential for intimal hyperplasia development.

PLoS One 2017 10;12(10):e0185820. Epub 2017 Oct 10.

Department of Surgery, Leiden University Medical Center (LUMC), Leiden, The Netherlands.

Objective: Genetic P300/CBP-associated factor (PCAF) variation affects restenosis-risk in patients. PCAF has lysine acetyltransferase activity and promotes nuclear factor kappa-beta (NFκB)-mediated inflammation, which drives post-interventional intimal hyperplasia development. We studied the contributing role of PCAF in post-interventional intimal hyperplasia.

Methods And Results: PCAF contribution to inflammation and intimal hyperplasia was assessed in leukocytes, macrophages and vascular smooth muscle cells (vSMCs) in vitro and in a mouse model for intimal hyperplasia, in which a cuff is placed around the femoral artery. PCAF deficiency downregulate CCL2, IL-6 and TNF-alpha expression, as demonstrated on cultured vSMCs, leukocytes and macrophages. PCAF KO mice showed a 71.8% reduction of vSMC-rich intimal hyperplasia, a 73.4% reduction of intima/media ratio and a 63.7% reduction of luminal stenosis after femoral artery cuff placement compared to wild type (WT) mice. The association of PCAF and vascular inflammation was further investigated using the potent natural PCAF inhibitor garcinol. Garcinol treatment reduced CCL2 and TNF-alpha expression, as demonstrated on cultured vSMCs and leukocytes. To assess the effect of garcinol treatment on vascular inflammation we used hypercholesterolemic ApoE*3-Leiden mice. After cuff placement, garcinol treatment resulted in reduced arterial leukocyte and macrophage adherence and infiltration after three days compared to untreated animals.

Conclusions: These results identify a vital role for the lysine acetyltransferase PCAF in the regulation of local inflammation after arterial injury and likely the subsequent vSMC proliferation, responsible for intimal hyperplasia.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185820PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5634597PMC
October 2017

Inhibition of 14q32 microRNA miR-495 reduces lesion formation, intimal hyperplasia and plasma cholesterol levels in experimental restenosis.

Atherosclerosis 2017 06 13;261:26-36. Epub 2017 Apr 13.

Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands; Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, The Netherlands. Electronic address:

Background And Aims: We aimed at investigating the role of 14q32 microRNAs in intimal hyperplasia and accelerated atherosclerosis; two major contributors to restenosis. Restenosis occurs regularly in patients treated for coronary artery disease and peripheral arterial disease. We have previously shown that inhibition of 14q32 microRNAs leads to increased post-ischemic neovascularization, and microRNA miR-494 also decreased atherosclerosis, while increasing plaque stability. We hypothesized that 14q32 microRNA inhibition has beneficial effects on intimal hyperplasia, as well as accelerated atherosclerosis.

Methods: Non-constrictive cuffs were placed around both femoral arteries of C57BL/6J mice to induce intimal hyperplasia. Accelerated atherosclerotic plaque formation was induced in hypercholesterolemic ApoE mice by placing semi-constrictive collars around both carotid arteries. 14q32 microRNAs miR-329, miR-494 and miR-495 were inhibited in vivo using Gene Silencing Oligonucleotides (GSOs).

Results: GSO-495 administration led to a 32% reduction of intimal hyperplasia. Moreover, the number of macrophages in the arterial wall of mice treated with GSO-495 was reduced by 55%. Inhibition of miR-329 and miR-494 had less profound effects on intimal hyperplasia. GSO-495 administration also decreased atherosclerotic plaque formation by 52% and plaques of GSO-495 treated animals showed a more stable phenotype. Finally, cholesterol levels were also decreased in GSO-495 treated animals, via reduction of the VLDL-fraction.

Conclusions: GSO-495 administration decreased our primary outcomes, namely intimal hyperplasia, and accelerated atherosclerosis. GSO-495 administration also favourably affected multiple secondary outcomes, including macrophage influx, plaque stability and total plasma cholesterol levels. We conclude that 14q32 microRNA miR-495 is a promising target for prevention of restenosis.
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http://dx.doi.org/10.1016/j.atherosclerosis.2017.04.011DOI Listing
June 2017

Inhibition of 14q32 MicroRNAs miR-329, miR-487b, miR-494, and miR-495 increases neovascularization and blood flow recovery after ischemia.

Circ Res 2014 Sep 1;115(8):696-708. Epub 2014 Aug 1.

From the Department of Surgery (S.M.J.W., A.J.N.M.B., R.C.M.d.J., M.R.d.V., E.A.B.P., M.C.B., P.H.A.Q., A.Y.N.) and Einthoven Laboratory for Experimental Vascular Medicine (S.M.J.W., A.J.N.M.B., R.C.M.d.J., M.R.d.V., E.A.B.P., P.H.A.Q., A.Y.N.), Leiden University Medical Center, Leiden, The Netherlands; Department of Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark (S.P.S.); and Idera Pharmaceuticals, Cambridge, MA (N.L.M., E.R.K.).

Rationale: Effective neovascularization is crucial for recovery after cardiovascular events.

Objective: Because microRNAs regulate expression of up to several hundred target genes, we set out to identify microRNAs that target genes in all pathways of the multifactorial neovascularization process. Using www.targetscan.org, we performed a reverse target prediction analysis on a set of 197 genes involved in neovascularization. We found enrichment of binding sites for 27 microRNAs in a single microRNA gene cluster. Microarray analyses showed upregulation of 14q32 microRNAs during neovascularization in mice after single femoral artery ligation.

Methods And Results: Gene silencing oligonucleotides (GSOs) were used to inhibit 4 14q32 microRNAs, miR-329, miR-487b, miR-494, and miR-495, 1 day before double femoral artery ligation. Blood flow recovery was followed by laser Doppler perfusion imaging. All 4 GSOs clearly improved blood flow recovery after ischemia. Mice treated with GSO-495 or GSO-329 showed increased perfusion already after 3 days (30% perfusion versus 15% in control), and those treated with GSO-329 showed a full recovery of perfusion after 7 days (versus 60% in control). Increased collateral artery diameters (arteriogenesis) were observed in adductor muscles of GSO-treated mice, as well as increased capillary densities (angiogenesis) in the ischemic soleus muscle. In vitro, treatment with GSOs led to increased sprout formation and increased arterial endothelial cell proliferation, as well as to increased arterial myofibroblast proliferation.

Conclusions: The 14q32 microRNA gene cluster is highly involved in neovascularization. Inhibition of 14q32 microRNAs miR-329, miR-487b, miR-494, and miR-495 provides a promising tool for future therapeutic neovascularization.
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http://dx.doi.org/10.1161/CIRCRESAHA.114.304747DOI Listing
September 2014

TLR4 accessory molecule RP105 (CD180) regulates monocyte-driven arteriogenesis in a murine hind limb ischemia model.

PLoS One 2014 19;9(6):e99882. Epub 2014 Jun 19.

Department of Surgery, Leiden University Medical Center, Leiden, the Netherlands; Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands.

Aims: We investigated the role of the TLR4-accessory molecule RP105 (CD180) in post-ischemic neovascularization, i.e. arteriogenesis and angiogenesis. TLR4-mediated activation of pro-inflammatory Ly6Chi monocytes is crucial for effective neovascularization. Immunohistochemical analyses revealed that RP105+ monocytes are present in the perivascular space of remodeling collateral arterioles. As RP105 inhibits TLR4 signaling, we hypothesized that RP105 deficiency would lead to an unrestrained TLR4-mediated inflammatory response and hence to enhanced blood flow recovery after ischemia.

Methods And Results: RP105-/- and wild type (WT) mice were subjected to hind limb ischemia and blood flow recovery was followed by Laser Doppler Perfusion Imaging. Surprisingly, we found that blood flow recovery was severely impaired in RP105-/- mice. Immunohistochemistry showed that arteriogenesis was reduced in these mice compared to the WT. However, both in vivo and ex vivo analyses showed that circulatory pro-arteriogenic Ly6Chi monocytes were more readily activated in RP105-/- mice. FACS analyses showed that Ly6Chi monocytes became activated and migrated to the affected muscle tissues in WT mice following induction of hind limb ischemia. Although Ly6Chi monocytes were readily activated in RP105-/- mice, migration into the ischemic tissues was hampered and instead, Ly6Chi monocytes accumulated in their storage compartments, bone marrow and spleen, in RP105-/- mice.

Conclusions: RP105 deficiency results in an unrestrained inflammatory response and monocyte over-activation, most likely due to the lack of TLR4 regulation. Inappropriate, premature systemic activation of pro-inflammatory Ly6Chi monocytes results in reduced infiltration of Ly6Chi monocytes in ischemic tissues and in impaired blood flow recovery.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0099882PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4063870PMC
October 2015
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