Immunolocalization of platelet-derived growth factor receptor-β (PDGFR-β) and pericytes in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)

Abstract

Aims: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is identified by aggregates of NOTCH3 extracellular domain (N3ECD) along capillaries and the deposition of granular osmiophilic material (GOM). We assessed the pattern of distribution of pericytes in relation to N3ECD deposits in cerebral microvessels of CADASIL subjects.

Methods: We assessed post mortem brains from (n = 50) subjects with CADASIL, cerebral small vessel disease, and similar-age cognitively normal and older controls. Immunohistochemical and immunofluorescent staining methods were used to study the distribution and quantify immunoreactivities of the platelet-derived growth factor receptor-β (PDGFR-β) (for pericytes) and microvascular markers in the frontal cortex and white matter.

Results: PDGFR-β antibody stained cells typical of pericytes in capillaries and small arterioles in both the grey and white matter. PDGFR-β reactive pericytes adopted 'crescent' morphology wrapped closely around capillary walls readily evident in cross-sections. We noted considerable overlap between PDGFR-β and N3ECD imunoreactivities in capillaries. Quantitative analysis of PDGFR-β immunoreactivity revealed significant differences in PDGFR-β %A in CADASIL compared with young controls (P < 0.05). PDGFR-β %A was further positively correlated with the basement membrane marker collagen IV (r = 0.529, P = 0.009), but was not associated with GLUT-1, the marker for endothelial cells.

Conclusions: Our results suggest increased expression of PDGFR-β immunoreactive pericytes in cerebral microvessels in CADASIL compared with similar age controls. While we cannot confirm whether PDGFR-β-expressing pericytes produce N3ECD and hence GOM, our findings demonstrate that up-regulation of pericyte-like cells is associated with microvascular changes, including loss of vascular smooth muscle cells in CADASIL.

Keywords: CADASIL; capillaries; dementia; pericyte; platelet-derived growth factor receptor; vascular dementia; vascular smooth muscle cell.

Figure 1

PDGFR‐β immunoreactivity in pericyte‐like cells around capillaries in the frontal white matter from a CADASIL subject. (A) Strong PDGFR‐β immunostaining in pericytes associated with capillaries within the cortex, with little light staining in neurons. (B) PDGFR‐β only stained capillaries in the white matter. (C) Immunofluorescent labelling with PDGFR‐β (green) and GLUT‐1 (red) antibodies. There was differential of the two markers without significant overlap along capillaries, indicating cell‐specific GLUT‐1 staining in endothelial cells, and PDGFR‐β in perivascular cells. (D) Higher magnification of outlined box in (C), demonstrating PDGFR‐β (green) immunostained pericytes, which appear as classic ‘crescent’ cell bodies with elongated processes stretching around the capillary wall. Haematoxylin counterstain in A and B. Magnification bar = 20 μm in A, B and D, and 50 μm in C. CADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; PDGFR‐β, platelet‐derived growth factor receptor‐β.

Figure 2

PDGFR‐β immunoreactivity in cerebral microvessels in CADASIL and cerebral SVD. Immunofluorescent labelling of PDGFR‐β (green), smooth muscle α‐actin (SMA; magenta) and GLUT‐1 (red) in capillaries in the frontal white matter, counterstained with DAPI. (A and B) Sections from a 68‐year‐old female CADASIL case with R133C mutation; (B and C), an 81‐year‐old female patient with sporadic SVD; (E and F), a 94‐year‐old nondemented female control. Severe capillary degeneration was observed in CADASIL (A and B), with GLUT‐1 expression observed in endothelial cells with blebs, compared with SVD (C and D) and control subject (E and F). There was minimal SMA immunoreactvity in pericytes (D, arrow) or capillaries. Magnification bar = 33.4 μm in A, 10.4 μm in B, C and D, 15 μm in E and 11.5 μm in F. CADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; PDGFR‐β, platelet‐derived growth factor receptor‐β; SVD, small vessel disease.

Figure 3

PDGFR‐β immunoreactivity in relation to N3ECD deposits, as a marker of GOM along capillaries in CADASIL. A and B, N3ECD immunostaining in micro‐deposits (GOM) around capillaries. Post mortem human brain tissue from a 68‐year‐old female CADASIL case with R133C mutation. (C) PDGFR‐β (green) immunostained pericyte in cross‐section. (D) N3ECD (red) accumulation around the same pericyte (as in C). (E) Composite of both PDGFR‐β (green) and NOTCH3ECD (red), demonstrating co‐localization for both antibodies around the pericyte. (F) Section of capillary with PDGFR‐β (green) and NOTCH3ECD (red) immunoreactivities, demonstrating abundance of N3ECD deposits (red) associated with the capillary wall. There is some overlap with pericytes within the tissue. Magnification bar = 20 μm in A and B, 11 μm in C–E, and 24 μm in F. CADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; GOM, granular osmiophilic material; PDGFR‐β, platelet‐derived growth factor receptor‐β.

Figure 4

PDGFR‐β immunoreactivity associated with arterioles and cerebral microvessels in CADASIL. (A–C) Immunohistochemical staining for PDGFR‐β (A), SMA (B) and N3ECD (C) in a partially hyalinized arteriole in the white matter of a 44‐year‐old female CADASIL subject with R153C mutation. (A) PDGFR‐β reactivity was observed in the endothelial cells of arterioles and expressed abluminally to VSMC (arrow) and in the adventitia. (B) SMA and N3ECD reactivities were predominantly within the tunica media of the same vessel (C). (D–F) PDGFR‐β immunostaining in 49‐year‐old cognitively normal control female, indicating diffuse PDGFR‐β staining within arteriolar walls (D). SMA staining revealed intact vascular smooth muscle cells (E) without presence of N3ECD aggregates (F). (G–I) Immunofluorescent labelling of PDGFR‐β (green), SMA (magenta) and GLUT‐1 (red) in arterioles of the frontal white matter, with DAPI counterstain. (G) Vascular smooth muscle cell (demonstrated by SMA, magenta) degeneration in arterioles in a 68‐year‐old female CADASIL case with R133C mutation, with increased PDGFR‐β (green) expression within the vessel wall. (H) Some PDGFR‐β (green) reactivity in the endothelial cell layer of arteriole from an 81‐year‐old female patient with sporadic SVD with mild VSMC degeneration compared with CADASIL (cf. D). (I) PDGFR‐β (green) expression was absent in an arteriole in a 94‐year‐old nondemented female control, although GLUT‐1 (red) and SMA (magenta) appeared normal. (J–L) Immunofluorescent labelling of PDGFR‐β (green), SMA (magenta) and N3ECD (red) in arterioles of white matter, confirming that N3ECD only accumulated in the tunica media in CADASIL (J), although PDGFR‐β (green) immunoreactivity was apparent in both arterioles from CADASIL (J) and SVD cases (K). (L) GLUT‐1 immunostaining in endothelial cells of arteriole of a control subject, whereas PDGFR‐β reactivity was meagre, if any. Scale bar = 50 μm in A–F, 40 μm in G, 27.5 μm in H, 47.5 μm in I, 52 μm in J, 57 μm in K and 49.5 μm in L. CADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; PDGFR‐β, platelet‐derived growth factor receptor‐β; SMA, smooth muscle alpha actin; SVD, small vessel disease; VSMC, vascular smooth muscle cell.

Figure 5

Immunofluorescent labelling of PDGFR‐β (green), SMA (magenta) and GLUT‐1 (red) in an arteriole within the frontal white matter of a 68‐year‐old female CADASIL subject with R133C mutation. Expression of GLUT‐1 (red) by endothelial cells was markedly diminished in this arteriole. However, there was marked PDGFR‐β (green) reactivity. Scale bar is 47.62 μm. CADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; PDGFR‐β, platelet‐derived growth factor receptor‐β; SMA, smooth muscle alpha actin.

Figure 6

Quantification of PDGFR‐β immunoreactivity in the frontal white matter in CADASIL. (A) Quantitative analysis of PDGFR‐β percent area (%A) revealed a trend for differences between the groups (anova, P = 0.084, n = 6–7 samples per group). However, post hoc tests revealed significant differences between CADASIL subjects and young controls, and the old control group compared with young controls (Tukey's test, *P < 0.05). (B) Lack of relationship between PDGFR‐β %A and GLUT‐1 immunoreactivity (r = 0.312, P = 0.148). (C) PDGFR‐β %A was significantly correlated with the basement membrane marker COL4 (r = 0.529, P = 0.009). CADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; PDGFR‐β, platelet‐derived growth factor receptor‐β.