Evidence for angiogenesis in Parkinson's disease, incidental Lewy body disease, and progressive supranuclear palsy

Abstract

Angiogenesis has not been extensively studied in Parkinson's disease (PD) despite being associated with other neurodegenerative disorders. Post-mortem human brain tissues were obtained from subjects with pathologically confirmed Parkinson's disease (PD) and progressive supranuclear palsy (PSP), a rapidly progressing Parkinsonian-like disorder. Tissues were also obtained from subjects with incidental Lewy body disease (iLBD) who had Lewy bodies in the substantia nigra pars compacta (SN(pc)) but had not been diagnosed with PD, and age-matched controls without Lewy body pathology. The SNpc, putamen, locus ceruleus (LC) and midfrontal cortex were examined for integrin αvβ3, a marker for angiogenesis, along with vessel number and activated microglia. All parkinsonian syndromes had greater αvβ3 in the LC and the SN(pc), while only PD and PSP subjects had elevated αvβ3 in the putamen compared to controls. PD and PSP subjects also had increases in microglia number and activation in the SN(pc) suggesting a link between inflammation and clinical disease. Microglia activation in iLBD subjects was limited to the LC, an area involved at an early stage of PD. Likewise, iLBD subjects did not differ from controls in αvβ3 staining in the putamen, a late area of involvement in PD. The presence of αvβ3 reactive vessels in PD and its syndromes is indicative of newly created vessels that have not likely developed the restrictive properties of the blood brain barrier. Such angiogenic vessels could contribute to neuroinflammation by failing to protect the parenchyma from peripheral immune cells and inflammatory or toxic factors in the peripheral circulation.

Fig 1. Integrin αvβ3 staining in post-mortem human brain tissue

Endothelial cells of human post-mortem brain tissue were labeled with mouse anti-human integrin αvβ3 antibody and visualized with the chromagen DAB. Integrin αvβ3 reactive vessels are shown in post-mortem tissues from non-pathological controls, incidental Lewy Body Disease (iLBD), Parkinson’s disease (PD) and progressive supranuclear palsy (PSP) subjects. Note the distinct pattern of staining along vessels. In most cases only a small portion of the longitudinal vessel is stained. In other cases the vessels are perpendicular resulting in a ring of staining. The grey cells seen in the SNpc are neuromelanin-containing cells that are evident in unstained tissues and could be distinguished from the αvβ3 staining by stain color (in the original color images), and by morphology. Black scale bars = 100 μm.

Fig 2. Intensity of αvβ3 staining in post-mortem human brain tissue samples

Integrin αvβ3 immunoreactivity was quantified as optical density (OD) of the vessels (see Methods). Integrin αvβ3 immunoreactivity was significantly greater in all Parkinsonian conditions compared with controls in the SN_pc, and locus ceruleus. In the putamen, only PD and PSP were different than control. In the Midfrontal Cortex, there was variability and none of the differences reached statistical significance. Data is expressed as Mean ± SEM. Statistical significance was determined for each area using ANOVA with Dunnett test for mean comparison to control with * (p<0.05) and ** (p<0.01).

Fig 3. Vessel number in post-mortem human tissue samples

Vessel numbers were counted under bright field microscopy as described in the Methods. PSP subjects had increased vessel number/mm² in the SN_pc and MFC. iLBD subjects had a small but significant increase in vessel number/mm² in the MFC. Vessel number/mm² appeared increased in the LC for all Parkinsonian disorders but did not reach statistical significance. Data was expressed as Mean ± SEM. Statistical significance was determined using ANOVA with Dunnett test for mean comparison to control with * (p<0.05) and ** (p<0.01).

Fig 4. Activated microglia in post-mortem human tissue samples

Activated microglia were labeled using an antibody to MHC class II antigen and visualized with the chromagen DAB. In the SNpc, there were neuromelanin-containing cells, as evident in the control condition. Microglia could be distinguished form neuromelanin containing cells by size and morphology. The black scale bar = 100 μm. Insets in the panels were taken at higher magnification and show microglia morphology typical of the microglia in that condition. White scale bars in the insets are 20 μm.

Fig. 5. Stereological assessment of Microglial density

Stereology was used to determine the number of activated microglia in a given region. Density was obtained by dividing total number of activated microglia by volume of interest (refer to Methods). The number of activated microglia was significant for PD in the SN_pc. PSP subjects showed an increase in both the SN_pc and the MFC. iLBD subjects had an increase in the LC. Data is expressed as Mean ± SEM. Statistical significance was determined using ANOVA with Dunnett test for mean comparison to control with * (p<0.05) and ** (p<0.01).