Lysyl hydroxylase 1 (LH1) deficiency promotes angiotensin II (Ang II)-induced dissecting abdominal aortic aneurysm

Abstract

Rationale: The progressive disruption of extracellular matrix (ECM) proteins, particularly early elastin fragmentation followed by abnormalities in collagen fibril organization, are key pathological processes that contribute to dissecting abdominal aortic aneurysm (AAA) pathogenesis. Lysyl hydroxylase 1 (LH1) is essential for type I/III collagen intermolecular crosslinking and stabilization. However, its function in dissecting AAA has not been explored. Here, we investigated whether LH1 is significantly implicated in dissecting AAA progression and therapeutic intervention. Methods and Results: Sixteen-week-old male LH1-deficient and wild-type (WT) mice on the C57Bl/6NCrl background were infused with angiotensin II (Ang II, 1000 ng/kg per minute) via subcutaneously implanted osmotic pumps for 4 weeks. Ang II increased LH1 levels in the abdominal aortas of WT mice, whereas mice lacking LH1 developed dissecting AAA. To evaluate the related mechanism, we performed whole-transcriptomic analysis, which demonstrated that LH1 deficiency aggravated gene transcription alterations; in particular, the expression of thrombospondin-1 was markedly upregulated in the aortas of LH1-deficient mice. Furthermore, targeting thrombospondin-1 with TAX2 strongly inhibited the proinflammatory process, matrix metalloproteinase (MMP) activity and vascular smooth muscle cells (VSMCs) apoptosis, ultimately decreasing the incidence of dissecting AAA. Restoration of LH1 protein expression in LH1-deficient mice by intraperitoneal injection of an adeno-associated virus normalized thrombospondin-1 levels, subsequently alleviating dissecting AAA formation and preserving aortic structure and function. Consistently, in human AAA specimens, decreased LH1 expression was associated with increased thrombospondin-1 levels. Conclusions: LH1 deficiency contributes to dissecting AAA pathogenesis, at least in part, by upregulating thrombospondin-1 expression, which subsequently enables proinflammatory processes, MMP activation and VSMCs apoptosis. Our study provides evidence that LH1 is a potential critical therapeutic target for AAA.

Keywords: angiotensin II; dissecting abdominal aortic aneurysm; lysyl hydroxylase; mice; thrombospondin-1.

Figure 1

Lysyl hydroxylase 1 (LH1) deficiency triggers dissecting abdominal aortic aneurysm (AAA) formation following 4 weeks of angiotensin II (Ang II) infusion. A, Representative morphology of the aortas of wild-type (WT) and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (Plod1)^-/- mice. B, The incidence of dissecting AAA and aortic aneurysm rupture in WT and Plod1^-/- mice in response to Ang II administration (n = 20). Two-sided fisher's exact test. C, Representative images of transabdominal ultrasound measurements (the lumen is indicated by the yellow line) at day 28 after saline or Ang II infusion. D, Quantification of the abdominal aorta diastolic diameter, pulse-wave velocity (PWV) and distensibility of the aortic wall by ultrasound at day 28 after saline or Ang II infusion (n = 10). E, Representative immunoblotting images of LH1 and quantitative analysis of LH1 expression in the abdominal aortas of WT and Plod1^-/- mice after Ang II or saline infusion (n = 6). ND indicates not detected. Student's unpaired two-tailed t-test. F, Representative immunostaining images of LH1 (red), elastin (green) and DAPI (blue) in the abdominal aorta. G, The systolic blood pressure of each group at day 28 after saline or Ang II infusion (n = 10). Two-way ANOVA followed by the Bonferroni post hoc test was used in D and G.

Figure 2

Lysyl hydroxylase 1 (LH1) deficiency increases structural damage to the aorta in response to angiotensin II (Ang II) administration. A, Representative cross-sections of hematoxylin and eosin (H&E)-, Verhoeff-Van Gieson (VVG)-, Masson trichrome- and picrosirius red (PSR)-stained abdominal aorta tissues from mice after 4 weeks of Ang II or saline infusion. Quantification of elastin breakage (VVG staining, B) and collagen content (PSR staining, C) (n = 6). D, Transmission electron microscopy images showing an overview, collagen fibers, and cross-sectioned fibers in the abdominal aortas of mice. Two-way ANOVA followed by Bonferroni post hoc test.

Figure 3

Lysyl hydroxylase 1 (LH1) deficiency aggravates inflammation, matrix metalloproteinase (MMP) activity and apoptosis of vascular smooth muscle cells (VSMCs) in the abdominal aorta. A, Cluster analysis heat map showing the transcript levels of differentially expressed genes (DEGs) in the abdominal aortas of wild-type (WT) and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (Plod1)^-/- mice following 2 weeks of saline or angiotensin II (Ang II) infusion. The color scale illustrates the relative expression levels across all samples: red represents an expression level above the mean, and blue represents an expression level lower than the mean. The dendrogram on the left of the heat map shows the clustering of the transcripts (n = 5). B, UpSet plot of the intersection of four groups. The histogram shows the number of DEGs in each subset. C, Human Phenotype Ontology analysis of DEGs between Plod1^-/- and WT mice after Ang II infusion. D, Top enriched terms, as identified by from Gene Ontology (GO) enrichment analysis of the DEGs between Plod1^-/- and WT mice after Ang II infusion. E, Representative immunofluorescence staining of macrophages in the abdominal aorta after 4 weeks of saline or Ang II infusion. The insets are higher magnification images. F, mRNA expression of the inflammatory cytokines Ccl2 and Il6 in the abdominal aorta (n = 6). G, Serum levels of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) were measured by ELISA (n = 6). H, Representative gelatin zymography of abdominal aortas from the indicated groups. I, Representative images and quantification of apoptotic cells (upper) and α-SMA expression (lower) in the abdominal aorta (n = 6). J, Volcano plot displaying DEGs between Plod1^-/- and WT mice after Ang II infusion. K, Representative immunoblotting images for thrombospondin-1 in the abdominal aortas of WT and Plod1^-/- mice. L, Representative immunostaining images of thrombospondin-1 (red), elastin (green) and DAPI (blue) in the abdominal aorta. Two-way ANOVA followed by Bonferroni post hoc test.

Figure 4

Thrombospondin-1 is the key mediator of dissecting abdominal aortic aneurysm (AAA) pathogenesis in the absence of lysyl hydroxylase 1 (LH1). A, Representative aortas from procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (Plod1)^-/- mice treated with scrambled peptide (scrb) or TAX2 followed by angiotensin II (Ang II) infusion for 4 weeks. B, Incidence of dissecting AAA and aortic aneurysm rupture in Plod1^-/- mice treated with scrb or TAX2 in response to Ang II (n = 20). Two-sided fisher's exact test. C, Representative images of transabdominal ultrasound measurements (the lumen is indicated by the yellow line). D, Quantification of the abdominal aorta diastolic diameter, pulse-wave velocity (PWV) and distensibility of the aortic wall by ultrasound (n = 10). Student's unpaired two-tailed t-test. E, Representative hematoxylin and eosin (H&E)-, Verhoeff-Van Gieson (VVG)-, and Masson trichrome-stained cross-sections of the abdominal aortas of mice. F, Representative immunofluorescence staining of macrophages in the abdominal aorta following Ang II infusion for 2 weeks. The insets show higher magnification images. G, Representative gelatin zymography of abdominal aortas from the indicated groups. H, Representative images of apoptotic cells (upper) and α-SMA expression (lower) in the abdominal aorta.

Figure 5

Correction of lysyl hydroxylase 1 (LH1) deficiency by adeno-associated virus (AAV)-based gene therapy prevents angiotensin II (Ang II)-induced dissecting abdominal aortic aneurysm (AAA) formation in procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (Plod1)^-/- mice. A, Representative immunoblot analysis and immunofluorescence images of LH1 expression in the abdominal aortas of Plod1^-/- mice treated with AAV-GFP or AAV-LH1. The insets show higher magnification images. B, Representative immunoblot analysis and immunofluorescent images of thrombospondin-1 expression in the abdominal aortas of Plod1^-/- mice treated with AAV-GFP or AAV-LH1 followed by Ang II infusion for 2 weeks (n = 6). Student's unpaired two-tailed t-test. C, Representative aortas from Plod1^-/- mice treated with AAV-GFP or AAV-LH1 followed by Ang II infusion for 4 weeks. D, Incidence of dissecting AAA and aortic aneurysm rupture in response to Ang II in Plod1^-/- mice treated with AAV-GFP or AAV-LH1 (n = 20). Two-sided fisher's exact test. E, Representative images of transabdominal ultrasound measurements (the lumen is indicated by the yellow line). F, Quantification of the abdominal aorta diastolic diameter, pulse-wave velocity (PWV) and distensibility of the aortic wall by ultrasound (n = 10). Student's unpaired two-tailed t-test. G, Representative hematoxylin and eosin (H&E)-, Verhoeff-Van Gieson (VVG)-, and Masson trichrome-stained cross-sections of the abdominal aortas of mice. H, Transmission electron microscopy images showing collagen fibers and cross-sectioned fibers in the abdominal aortas of mice.

Figure 6

Lysyl hydroxylase 1 (LH1) are significantly reduced and thrombospondin-1 levels are elevated in specimens from humans with abdominal aortic aneurysm (AAA). Representative immunofluorescence images and quantification of LH1 (A) and thrombospondin-1 (B) expression. The boxed regions in the images are shown at a higher magnification on the right (n = 6). AU indicates arbitrary units. Student's unpaired two-tailed t-test.