HSP60 knockdown exerts differential response in endothelial cells and monocyte derived macrophages during atherogenic transformation

Abstract

Ectopic expression of HSP60 in vascular cells is known to activate auto-immune response that is critical to atherogenic initiation. However, the pathogenic relevance of the aberrant HSP60 upregulation in intracellular signaling pathways associated with atherogenic consequences in vascular cells remains unclear. The aim of the present study was to determine the role of endogenous HSP60 in atherogenic transformation of endothelial cells and macrophages. After generating primary evidence of oxidized low density lipoprotein (OxLDL) induced HSP60 upregulation in human umbilical vein endothelial cells (HUVEC), its physiological relevance in high fat high fructose (HFHF) induced early atherogenic remodelling was investigated in C57BL/6J mice. Prominent HSP60 expression was recorded in tunica intima and media of thoracic aorta that showed hypertrophy, lumen dilation, elastin fragmentation and collagen deposition. Further, HSP60 overexpression was found to be prerequisite for its surface localization and secretion in HUVEC. eNOS downregulation and MCP-1, VCAM-1 and ICAM-1 upregulation with subsequent macrophage accumulation provided compelling evidences on HFHF induced endothelial dysfunction and activation that were also observed in OxLDL treated- and HSP60 overexpressing-HUVEC. OxLDL induced concomitant reduction in NO production and monocyte adhesion were prevented by HSP60 knockdown, implying towards HSP60 mediated possible regulation of the said genes. OxLDL induced HSP60 upregulation and secretion was also recorded in THP-1 derived macrophages (TDMs). HSP60 knockdown in TDMs accounted for higher OxLDL accumulation that correlated with altered scavenger receptors (SR-A1, CD36 and SR-B1) expression further culminating in M1 polarization. Collectively, the results highlight HSP60 upregulation as a critical vascular alteration that exerts differential regulatory role in atherogenic transformation of endothelial cells and macrophages.

Figure 1

HSP60 expression in OxLDL treated HUVEC. Cells were treated with OxLDL (80 µg/ml) for 24 h and expression of HSP60 was evaluated by (a) western blotting followed by (b) its densitometric analysis and (c) immunocytochemistry followed by (d) its quantification by Image J analysis. Scale bar = 20 µm. CTCF corrected total cell fluorescence. Data were expressed as Mean ± SEM (n = 3). *p < 0.05, **p < 0.01 vs untreated control, unpaired two-tailed Student’s t-test.

Figure 2

Pro-atherogenic remodelling and HSP60 expression in thoracic aorta of HFHF diet fed mice. (a) Histological analysis of thoracic aortas stained with H × E (100× and 400×) were subjected to quantification of (b) Intima-media thickness (IMT) (n = 3; Scale bar = 100 µm) and (c) lumen area (n = 3). The sections were also subjected to (d) elastin autofluorescence analysis (upper lane, red arrows indicate elastin breaks; Scale bar = 100 µm) and collagen staining by picrosirius red (lower lane; Scale bar = 50 µm). The graphs represents (e) elastin fragmentation (n = 3), (f) collagen content (n = 5 for control, n = 4 for HFHF) and (g) collagen-to-elastin ratio (n = 4). The expression of HSP60 was analysed by (h) western blotting followed by (i) densitometry (n = 3) and (j) immunohistochemistry (Scale bar = 50 µm; arrows indicate HSP60⁺ stained areas) followed by (k) quantification (n = 3) by Image J. Data were expressed as Mean ± SEM. *p < 0.05, ***p < 0.001 vs Chow diet fed mice, unpaired two-tailed Student’s t-test. L lumen.

Figure 3

Surface localization and secretion of HSP60 in HUVEC. (a) OxLDL treated and pcDNA-HSP60 HUVECs were surface immunostained for HSP60 by following 1% PFA fixation protocol. Representative images are shown with HSP60 (A488), β-actin (A568) and nuclei (Hoechst). Scale bar = 100 µm. (b) The fluorescence was quantified using Image J. CTCF corrected total cell fluorescence (n = 4 for control, n = 3 for OxLDL and pcDNA-HSP60). **p < 0.01, ***p < 0.001 vs untreated control, One way ANOVA followed by Tukey’s multiple comparison test. Further, the transient overexpression of HSP60 in HSP60-GFP cells was assessed by (c) quantitative RT-PCR and (d) western blotting (n = 3). ***p < 0.001 vs Vector, unpaired two-tailed Student’s t-test. (e) The levels of HSP60 in conditioned media from HUVEC were analysed by ELISA (n = 3). Data were represented as Mean ± SEM. *p < 0.05, **p < 0.01 vs untreated control, ns non-significant, One way ANOVA followed by Tukey’s multiple comparison test.

Figure 4

Endothelial dysfunction and activation in thoracic aorta of HFHF diet fed mice. Thoracic aorta from Control and HFHF groups were subjected to quantitative RT-PCR for (a) eNOS, (b) MCP-1, (c) VCAM-1 and (d) ICAM-1 (n = 3). (e) Sections were immunostained for CD68 (Scale bar = 50 µm; arrows indicate CD68⁺ staining). (f) Quantification of the positively stained areas was carried out using Image J (n = 5). Data were expressed as Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs untreated control, unpaired two-tailed Student’s t-test. L lumen.

Figure 5

HSP60 mediated endothelial dysfunction in HUVEC. (a) Knockdown of HSP60 in HUVEC was confirmed by quantitative RT-PCR and western blotting. *p < 0.05, **p < 0.01, ***p < 0.001 vs KD-control, unpaired two-tailed Student’s t-test. (b) NO production from HUVEC was assessed using Griess’ reagent and (c) expression of eNOS mRNA was analysed by quantitative RT-PCR. Data were expressed as Mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs untreated control, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs OxLDL, and ⁺p < 0.05, ⁺⁺⁺p < 0.001 vs HSP60-GFP, One way-ANOVA followed by Tukey’s multiple comparison test.

Figure 6

HSP60 mediated endothelial activation in HUVEC. The adhesion of THP-1 cells to HUVEC subjected to various experimental conditions was assessed by (a) microscopic analysis and (b) the % monocyte adhesion was calculated relative to number of HUVECs (n = 5). Scale bar = 50 µm. **p < 0.01, ***p < 0.001 vs untreated control, ^##p < 0.01, ^###p < 0.001 vs OxLDL, and ⁺⁺⁺p < 0.001 vs HSP60-GFP, One way-ANOVA followed by Tukey’s multiple comparison test. (c) mRNA of MCP-1, VCAM-1 and ICAM-1 was assessed by quantitative RT-PCR (n = 3). Data were expressed as Mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs untreated control, ^##p < 0.01, ^###p < 0.001 vs OxLDL, and ⁺⁺⁺p < 0.001 vs HSP60-GFP, Two way-ANOVA followed by Tukey’s multiple comparison test.

Figure 7

Expression and secretion of HSP60 in OxLDL treated TDMs. TDMs were treated with OxLDL and expression of HSP60 was evaluated by (a) western blotting followed by (b) densitometry. (c) HSP60 secretion was checked in conditioned media by ELISA. Data were expressed as Mean ± SEM (n = 3). *p < 0.05, **p < 0.01, vs untreated control, unpaired two-tailed Student’s t-test.

Figure 8

OxLDL uptake and scavenger receptor expression in HSP60 KD TDMs. Knockdown of HSP60 in TDMs was confirmed by (a) quantitative RT-PCR and (b) western blotting (n = 3). **p < 0.01, vs untreated KD-Control, two-tailed Student’s t-test. TDMs were subjected to ORO staining to check the OxLDL uptake (c). Representative images of stained cells (Scale bar = 10 µm) and (d) quantitative measurement of OxLDL accumulation. mRNA expression of (e) SR-A1, (f) CD36 and (g) SR-B1 was evaluated by quantitative RT-PCR. (h) Comparative analysis of mRNA expression of the three SRs in OxLDL treated KD-Control and HSP60 KD TDMs expressed as percentage of total fold change observed in respective groups. Data were expressed as Mean ± SEM (n = 3). *p < 0.05, ***p < 0.001 vs untreated KD-Control. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs untreated HSP60 KD. ⁺p < 0.05, ⁺⁺⁺p < 0.001 KD-Control group vs HSP60 KD group, ns non-significant, Two-way ANOVA followed by Tukey’s multiple comparison test.

Figure 9

OxLDL induced polarization of HSP60 KD TDMs. OxLDL induced polarization events were checked by assessing the mRNA expression of M1 markers (a) iNOS and (b) IL-6 and M2 markers (c) ARG-1 and (d) IL-10 in OxLDL treated KD-Control and HSP60 KD TDMs. Data were expressed as Mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs untreated KD-Control. ^###p < 0.001 vs untreated HSP60 KD. ⁺p < 0.05, ⁺⁺⁺p < 0.001 KD-Control group vs HSP60 KD group, ns non-significant, Two-way ANOVA followed by Tukey’s multiple comparison test.