Table 1.
Primer sequences against human cDNA sequences used for qRT-PCR.
Sequences are listed in the 5’ to 3’ orientation.
Fig 1.
NOTCH3 protein localization in CADASIL and control arteries.
Localization of NOTCH3 fragmentation products was performed in CADASIL (top row) and non-CADASIL frontal cortex by immunohistochemistry with UMI-F, which recognizes the neo-epitope generated by fragmentation of NOTCH3 at the junction between EGF-like repeats 1 and 2. The left panels show leptomeningeal arteries (LM) and the right panels show white matter arteries (WM). Arteries from both regions demonstrate medial staining corresponding to the vascular smooth muscle layer of arteries with deficiency in the intimal and adventitial layers. Scale bar shows 100 microns.
Fig 2.
Vascular smooth muscle cell proteins in CADASIL and control arteries.
Eight antibodies (labeled on the left) were used for immunohistochemical localization of respective smooth muscle antigens in CADASIL and control brains. Both leptomeningeal (LM) and white matter (WM) vessels are shown to illustrate representative positively-stained vessels. Scale bar shows 100 microns.
Fig 3.
Vascular smooth muscle cell proteins in CADASIL and control arteries.
Eight antibodies (labeled on the left) were used for immunohistochemical localization of respective smooth muscle antigens in CADASIL and control brains. Both leptomeningeal (LM) and white matter (WM) vessels are shown to illustrate representative positively-stained vessels. Scale bar shows 100 microns.
Fig 4.
Markers exhibiting differential levels of staining between CADASIL and control arteries or between leptomeninges and white matter.
Images from Figs 2 and 3 are placed side by side for easier comparison. A) Immunohistochemical examination of markers (labels to the left) which had decreased staining in CADASIL relative to controls. Representative leptomeningeal arteries (LM) are shown. B) Immunohistochemical comparison of markers (labeled on the left) in leptomeningeal arteries and white matter arteries (WM) in representative CADASIL brain samples. A select group of markers showed moderate white matter enrichment pattern in CADASIL vessels. Scale bar shows 100 microns.
Fig 5.
Quantification of mRNA of 16 markers in CADASIL and control brain.
RNA from frozen frontal lobe of CADASIL and control brains were analyzed by qRT-PCR; expression values were normalized to 18S RNA and then controls were set to 1 (n = 8 per group; 37% female; average age at death was 62 years for controls and 64 years for CADASIL). X-axis show fold changes relative to controls. Significant differences with a p<0.05 are denoted by asterisks.
Table 2.
Antibodies against proteins used in phase 2 screen for NOTCH3-like vascular markers in CADASIL.
Protein targets are listed alongside antibody catalogue numbers as annotated in the Human Protein Atlas or listed in the Developmental Studies Hybridoma Bank. The presence of a NOTCH3 staining pattern is listed (defined as predominantly medial and enriched in CADASIL arteries). *Two antibodies were delisted from the Human Protein Atlas since the initiation of the project.
Fig 6.
Localization of CD63 and CTSH in CADASIL cerebral arteries.
Immunohistochemical localization was performed for the N-terminal fragment (NTF; recognized by UMI-F), CD63 (H5C6), and CTSH on serial sections of CADASIL frontal lobe. Both leptomeningeal (LM) and white matter (WM) arteries showed medial staining which exceeded levels in the adventitia and intima. Scale bar shows 100 microns.
Fig 7.
Image analysis workflow depicting VesSeg.
A leptomeningeal vessel stained for NOTCH3 was used to demonstrate the process (top panel). The artery was pre-processed using sequential color deconvolution to remove the hematoxylin signal and to focus on the DAB signal (second panel). Masking and then cropping were performed on the processed image to limit analysis to the vessel wall. Spokes were generated for the vessel, resulting in radial segmentation of the wall (third panel). Furthermore, stained regions were thresholded using a custom criterion for processing (fourth vessel). Finally, a histogram depicting the frequency of staining along the vessel wall width was generated (higher frequency corresponds to increased level of staining in that region; last panel).
Fig 8.
Quantitative analysis of marker deposition in CADASIL arteries using VesSeg.
A) Mean staining distribution patterns plotted for each marker. The wall of each vessel was first split into 100 circumferential bands, and then the average staining for each region is calculated. Blue represents the marker distribution, while black indicates the reference NOTCH3 distribution. B) The staining scores for the intimal, medial, and adventitial layers of the blood vessel wall plotted by antibody. After pre-processing and segmentation, signal density maps were divided into three zones corresponding to adventitia, media, and intima. Then, the average pixel value in each layer was calculated and then normalized by subtracting the average value for the entire vessel from it. The normalized score for each layer was plotted along with standard deviation of all the vessels analyzed per marker. Red arrows show proteins which have increased expression in the media of arteries. Significant differences between the tunica intima and media staining is indicated by an asterisk; p < 0.05. C) Similarity scores between each marker and the reference NOTCH3 and ADIRF distributions. The score was calculated as the inverse Jensen-Shannon distance. D) t-SNE plots of vessel wall feature representations comparing each antibody (in black) to NOTCH3 (in blue). We used 100-dimensional vectors corresponding to the staining distribution of tangential sections of the blood vessel analysis to determine how the antibody-stained vessels are represented by our image analysis algorithm using t-distributed stochastic neighbor embedding (t-SNE). Black boxes highlight the t-SNE plot comparisons between markers with expression in the media of vessels (CD63 and CTSH). The final panel is a t-SNE plot comparing the vessel wall feature representations for NOTCH3, CTSH, and CD63 displayed together.
Fig 9.
In situ mRNA localization and levels of expression of non-myocyte factors, CD63 and CTSH, in CADASIL arteries.
(A) Representative artery which display clear CD63 mRNA signal in the media of leptomeningeal arteries by in situ hybridization. Similar findings were obtained with multiple vessels in several patients. Arrows point to the pink signal of mRNA molecule in vascular smooth muscle layer of arteries. Scale bar shows 100 microns. (B) Quantitative RT-PCR was performed for CD63 and CTSH to assess relative expression of corresponding mRNA in CADASIL and control frontal brain samples.
Fig 10.
Proximity of CD63 and NOTCH3 fragmentation products in CADASIL.
Experiments were performed to assess whether NOTCH3 fragmentation product NTF (recognized by UMI-F antibody) is in close association with CD63 (recognized by H5C6) in tissue. Arrows point to the magenta signal indicating positive PLA signal in the media, in the vicinity of vascular smooth muscle cells. Leptomeningeal (LM) and white matter (WM) arteries are shown. The left side shows 400X images; the right side shows 1000X images of a subregion of the same vessels. Scale bar shows 100 microns in the left panels.
Fig 11.
Cellular origin of markers localized in CADASIL vessels.
Cell transcriptomic analysis of proteins that accumulate in CADASIL media and of SMC markers. Data was obtained from publicly accessible sets described in [34], in which mouse vessel single cell transcriptomic analysis was performed. All values shown in the heatmap represent fraction of expression normalized to the total number of counts reported across all cell types examined. Marker names are arrayed on the y-axis and cell types along the x-axis. The markers were grouped to cluster non-canonical SMC markers (NC SMC), newly identified NOTCH3 distribution proteins (N3-like; CD63 and CTSH) with NOTCH3, previously identified molecules that were identified in CADASIL vessels [21] (CADASIL-enriched), and canonical SMC markers (C SMC).