Endothelial primary cilia inhibit atherosclerosis

Abstract

Primary cilia are microtubule-based structures present on most mammalian cells that are important for intercellular signaling. Cilia are present on a subset of endothelial cells where they project into the vessel lumen and are implicated as mechanical sensors of blood flow. To test the in vivo role of endothelial cilia, we conditionally deleted Ift88, a gene required for ciliogenesis, in endothelial cells of mice. We found that endothelial primary cilia were dispensable for mammalian vascular development. Cilia were not uniformly distributed in the mouse aorta, but were enriched at vascular branch points and sites of high curvature. These same sites are predisposed to the development of atherosclerotic plaques, prompting us to investigate whether cilia participate in atherosclerosis. Removing endothelial cilia increased atherosclerosis in Apoe(-/-) mice fed a high-fat, high-cholesterol diet, indicating that cilia protect against atherosclerosis. Removing endothelial cilia increased inflammatory gene expression and decreased eNOS activity, indicating that endothelial cilia inhibit pro-atherosclerotic signaling in the aorta.

Figure 1. Endothelial primary cilia are non‐uniformly distributed in the adult mouse aorta and are dispensable for development

1. A, B

   Primary cilia are enriched at branch points and the lesser curvature of the aorta of wild‐type mice, schematized in (A) and quantified in (B). The color in (A) indicates which side of the aorta is quantified in (B). Numbers indicate the region of the aorta (A) whose proportion of ciliated cells is quantified in (B). Error bars are ± 1 SEM and n = 7 mice.

2. C

   Aortic endothelial cilia are ablated in Tek‐Cre Ift88 ^C/− mice. Endothelial cells and cilia are labeled by staining for Pecam1 and Arl13b, respectively. Scale bars are 10 μm.

Figure EV1. Tek‐Cre mediates early, robust, and specific recombination in angioblasts

1. A–D

   Tek‐Cre is active in the mouse embryo in mTmG reporter mice by (A) E7.5 and labeling persists at (B) E8.5, (C) E10.5, and (D) in the aorta of adult mice. The arrowheads in (A) indicate the initial site of recombination in extraembryonic mesoderm. The asterisk in (B) indicates the yolk sac.

2. E, F

   Primary cilia are efficiently removed from aortic endothelial cells by Tek‐Cre‐mediated removal of Ift88 at (E) P5 and (F) P14.

3. G

   Primary cilia can also be ablated by Tek‐Cre‐mediated deletion of Kif3a. Endothelial cells and cilia are labeled by anti‐Pecam1 staining (green) and anti‐Arl13b staining (red).

Data information: Scale bars are 200 μm (A–C) and 10 μm (D–G). The arrowheads in (E–G) indicate primary cilia.

Figure EV2. Tek‐Cre‐mediated depletion of Ift88, Kif3a, Smo, or Kif3a in a Pecam ^Gt/Gt background does not affect ratios at weaning, whereas Tek‐Cre Pkd2 mice are subviable

1. Mice from the indicated crosses were weaned and genotyped at P21. Statistics (test indicated in table) suggest only Pkd2 endothelial cell deletion results in subviability.

2. Littermate Tek‐Cre Kif3a Pecam mice of the indicated genotypes were sacrificed at P604 and stained for Pecam1 and eNOS (in different fields). Loss of Pecam1, EC cilia, or both did not affect aortic EC eNOS expression. Scale bars, 10 μm.

Figure 2. Retinal angiogenesis and aortic endothelial polarity are unaffected in mice lacking EC cilia

1. The vasculature in P5 retinas from (A) mTmG reporter mice with and without EC cilia was visualized by EGFP fluorescence.

2. The fractional distance migrated to the edge of the retina was quantified. There was no statistically significant difference (P = 0.305, Student's two‐tailed t‐test, n = 6 mice for each group). The average value of 8 measurements (4 retina leaflets measured per eye on both eyes) was used for each mouse.

3. The number of vascular branch points was counted. There was no statistically significant difference (P = 0.25, Student's two‐tailed t‐test, n = 6 for control and n = 5 for mutant).

4. Control and mutant adult mouse aortas were stained for GM130 (green) to mark the Golgi, CD144 (red) to mark ECs, and DAPI (blue) to label DNA.

5. The angle between the Golgi, nucleus, and tissue axis was measured and binned as “left”, “right”, “upstream”, or “downstream”.

6. Six areas in the aorta were analyzed.

7. There was no difference in Golgi distribution at any single region or overall, nor was there a difference in the variance of the individual unbinned angles.

Data information: Error bars are +1 SEM. Scale bars are 500 μm. In (G), n = 3 control and mutant mice for each region except region 2 where n = 3 control and n = 2 mutant mice.

Figure 3. Loss of EC cilia accelerates atherosclerosis

1. A

   Scheme for induction of atherosclerosis Apoe ^−/− mice lacking Ift88 in the Tek‐Cre and Mx1‐Cre lineages (and control littermates). Following weaning, mice were placed on a high‐fat, high‐cholesterol diet for 8 weeks prior to analysis.

2. B

   Loss of EC cilia by Tek‐Cre increases atherosclerosis in the aortas of Apoe ^−/− mice on a high‐fat, high‐cholesterol diet as visualized by oil red O (ORO) staining (left panels), whereas loss of cilia genes in blood by Mx1‐Cre has no effect (right panels).

3. C

   Cropped views of the boxed areas in (B) showing increased atherosclerosis in mice with Tek‐Cre‐ but not Mx1‐Cre‐mediated loss of Ift88 in the aortic arch.

4. D–G

   Scatter plots of the percent ORO‐positive area of the thoracic aorta in (D, E) Tek‐Cre and (F, G) Mx1‐Cre mice, separated by (D, F) females and (E, G) males. Tek‐Cre Apoe ^−/− Ift88 ^C/− mice had a 58.9% (P = 0.0135, Student's two‐tailed t‐test, n = 13 control and n = 10 experimental mice) and 67% (P = 0.0106, Student's two‐tailed t‐test, n = 17 control and n = 14 experimental mice) increase in ORO‐positive area versus control littermates in females and males, respectively. Mx1‐Cre Apoe ^−/− Ift88 ^C/− mice had no significant difference in ORO‐positive area in females (P = 0.6643, Student's two‐tailed t‐test, n = 7 control and experimental mice) or males (P = 0.9381, Student's two‐tailed t‐test, n = 9 control and n = 7 experimental mice). Black bars represent the mean and error bars are ± 1 SEM. Aortas depicted in (B, C) are red in the scatter plots.

Figure EV3. Apoe ^−/− mice lacking EC cilia show increased atherosclerosis at the aortic sinus

1. The aortic sinuses from a cohort of male Apoe ^−/− mice with and without EC cilia were analyzed histologically with the indicated stains to determine plaque size and composition.

2. Quantitation of the ORO⁺ lesion area showed a trend for increased lesion size in mice lacking EC cilia (88% increase over controls, P = 0.1338, Student's two‐tailed t‐test, n = 5 mice each genotype). Black bars represent the mean and error bars are ± 1 SEM. Data points for sinuses in (A) are indicated in red.

3. The aortic sinuses of female Apoe ^−/− mice with and without EC cilia were stained for CD68 and smooth muscle actin (SMA) following 12 weeks of high‐fat, high‐cholesterol diet. Scale bars, 250 μm.

4. Quantitation of plaque composition from (C) showed no statistically significant differences between mice with and without EC cilia, although there was a trend for increased macrophage (CD68⁺) area. Black bars represent the mean and error bars are ± 1 SEM. Data points for sinuses in (C) are indicated in red. Student's two‐tailed t‐test, n = 5 control and n = 4 mutant mice.

Figure EV4. Extrahematopoietic, but not blood recombination, differs between Tek‐Cre and Mx1‐Cre mice and loss of Kif3a via Mx1‐Cre does not affect atherosclerosis

1. A, B

   Tail and peripheral blood DNA was isolated from adult Ift88 mice of the indicated genotypes carrying either (A) Tek‐Cre or (B) Mx1‐Cre. Genotyping for Ift88 indicated total loss of the conditional allele in blood from both Cre lines. However, only Tek‐Cre caused noticeable recombination in tail DNA, reflecting vascular recombination in the Tek‐Cre but not Mx1‐Cre lines. In each panel, the same 3 mice were assayed for blood and tail recombination and are presented in the same order. “C” indicates conditional allele, “+” indicates the wild‐type allele, and “‐” indicates the recombined null allele. The listed genotypes are the germ line genotypes of each mouse.

2. C

   Atherosclerosis was induced in control (Mx1‐Cre Kif3a ^C/+ Apoe ^−/−, Kif3a ^C/+ Apoe ^−/−, Kif3a ^C/− Apoe ^−/−) and mutant (Mx1‐Cre Kif3a ^C/− Apoe ^−/−) mice and the percent atherosclerosis assessed by oil red O staining. No difference was observed between control and experimental mice in males (P = 0.4692, Student's two‐tailed t‐test, n = 11 control and n = 4 experimental mice) or females (P = 0.5653, Student's two‐tailed t‐test, n = 8 control and n = 5 experimental mice). Horizontal bars are the mean and error bars are ± 1 SEM.

Figure 4. Loss of EC cilia activates pro‐atherosclerotic pathways in Apoe ^−/− mice

1. Transcripts for the inflammatory cytokines Il1b, Il6, Tnf1, and Tgfb1, the adhesion molecules Vcam1 and Sele, the lymphocyte marker Nos2 and macrophage marker Cd68, and the NFκB responsive genes Tnfaip3 and Hmox1 were upregulated in aortas from Apoe ^−/− mice lacking EC cilia, as assessed by RT–qPCR, while transcripts of the Hh target genes Gli1, Ptc1, and Ptc2 were not. Error bars are +1 SEM. n = 8 control and n = 10 mutant mice of mixed sex. P‐values calculated by Student's two‐tailed t‐test.

2. Pkd2 localizes to EC cilia in vivo. Adult mouse aortas were stained for Pkd2, acetylated α‐tubulin, and Hoechst to label DNA. Pkd2 localized strongly to primary cilia but also to other membranous structures in endothelial cells. Arrowheads indicate cilia. Representative cilium highlighted in inset. Scale bars, 10 μm.

3. Phosphorylation at serine 1,176 of eNOS was reduced in the aortas of mice lacking EC cilia. Aortas from control and mutant mice were stained for eNOS, p‐eNOS‐S1176, acetylated α‐tubulin, and Hoechst to label DNA. Arrowheads point out cilia. Arrowheads indicate cilia. Scale bars, 10 μm.

4. The ratio of the total fluorescence p‐eNOS‐S1176 to total eNOS in the upper thoracic aorta (near the first intercostal artery) was calculated for control and mutant mice. The relative p‐eNOS‐S1176 to eNOS ratio was reduced in the mutant to 72.7% the level of control siblings (P = 0.009, paired two‐tailed ratio t‐test, n = 7 sets of mice). Error bar is +1 SEM. Four sets consisted of one control mouse and one mutant mouse. Three sets consisted of two control or mutant mice and the average value of the two was used.

Figure EV5. Serum lipid profiles and body weight are unchanged by loss of EC cilia in Apoe ^−/− mice

1. Serum levels of cholesterol, HDL, LDL, triglyceride, and non‐esterified fatty acids were measured in n = 5 control (Tek‐Cre Ift88 ^C/+ Apoe ^−/−, Ift88 ^C/+ Apoe ^−/−, Ift88 ^C/− Apoe ^−/−) and n = 7 experimental (Tek‐Cre Ift88 ^C/− Apoe ^−/−) female mice. Large horizontal bars are the mean and error bars are ± 1 SEM.

2. There is no difference in body weight in Apoe ^−/− mice lacking EC cilia among males (P = 0.8911, Kolmogorov–Smirnov test, n = 46 control and n = 39 mice) or females (P = 0.9061, Kolmogorov–Smirnov test, n = 55 control and n = 52 mutant mice). These data include mice used for other experiments, hence the increased n. Horizontal bars are the mean and error bars are ± 1 SEM.