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. 2014 Sep;34(9):1472-82.
doi: 10.1038/jcbfm.2014.102. Epub 2014 May 28.

Ablation of MMP9 gene ameliorates paracellular permeability and fibrinogen-amyloid beta complex formation during hyperhomocysteinemia

Nino Muradashvili  1 , Reeta Tyagi  1 , Naira Metreveli  1 , Suresh C Tyagi  1 , David Lominadze  1
Affiliations

Ablation of MMP9 gene ameliorates paracellular permeability and fibrinogen-amyloid beta complex formation during hyperhomocysteinemia

Nino Muradashvili et al. J Cereb Blood Flow Metab. 2014 Sep.

Abstract

Increased blood level of homocysteine (Hcy), called hyperhomocysteinemia (HHcy) accompanies many cognitive disorders including Alzheimer's disease. We hypothesized that HHcy-enhanced cerebrovascular permeability occurs via activation of matrix metalloproteinase-9 (MMP9) and leads to an increased formation of fibrinogen-β-amyloid (Fg-Aβ) complex. Cerebrovascular permeability changes were assessed in C57BL/6J (wild type, WT), cystathionine-β-synthase heterozygote (Cbs+/-, a genetic model of HHcy), MMP9 gene knockout (Mmp9-/-), and Cbs and Mmp9 double knockout (Cbs+/-/Mmp9-/-) mice using a dual-tracer probing method. Expression of vascular endothelial cadherin (VE-cadherin) and Fg-Aβ complex formation was assessed in mouse brain cryosections by immunohistochemistry. Short-term memory of mice was assessed with a novel object recognition test. The cerebrovascular permeability in Cbs+/- mice was increased via mainly the paracellular transport pathway. VE-cadherin expression was the lowest and Fg-Aβ complex formation was the highest along with the diminished short-term memory in Cbs+/- mice. These effects of HHcy were ameliorated in Cbs+/-/Mmp9-/- mice. Thus, HHcy causes activation of MMP9 increasing cerebrovascular permeability by downregulation of VE-cadherin resulting in an enhanced formation of Fg-Aβ complex that can be associated with loss of memory. These data may lead to the identification of new targets for therapeutic intervention that can modulate HHcy-induced cerebrovascular permeability and resultant pathologies.

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Figures

Figure 1
Figure 1
Hyperhomocysteinemia (HHcy)-induced leakage of mouse pial venules. A dual-tracer probing method was used to define prevailing role of paracellular versus transcellular transport in pial venules of WT, Cbs gene knockout heterozygous (Cbs+/−), MMP9 gene knockout (Mmp9−/−), and Cbs+/− and Mmp9−/− double knockout (Cbs+/−/Mmp9−/−) mice. (A) Examples of images recorded immediately (baseline) and after 1 hour after infusion of fluorescein isothiocyanate (FITC) (green) and Alexa-647- conjugated bovine serum albumin (BSA) (red) tracers. Microvascular permeability was assessed by comparison of ratios of fluorescence intensities of dyes measured along the line profile probe (LPP) outside to that of inside of the vessel shown on images. Summary of changes in LLP ratios of fluorescence intensity values of FITC (B) and BSA-Alexa Fluor-647 (C) tracers. *P<0.05, versus WT; n=8 for all groups. Inset: Genotyping of Cbs and Mmp9 gene double knockout (Cbs+/−/Mmp9−/−) mice. Dual polymerase chain reaction (PCR) products suggest the heterozygous mutation of Cbs gene (Cbs+/−) and single PCR product suggests homozygous mutation of Mmp9 gene (Mmp9−/−) while their absence represents FVB or wild-type (C57BL) alleles.
Figure 2
Figure 2
Expression of vascular endothelial cadherin (VE-cadherin) in mouse brain vessels. (A) Examples of vessel images in samples obtained from wild type (WT), Cbs+/−, Cbs+/−, and Mmp9−/− double knockout (Cbs+/−/Mmp9−/−), and MMP9 gene knockout (Mmp9−/−) mice. VE-cadherin expression was assessed by fluorescence intensity along the vascular segment. Expression of VE-cadherin (red) in cortical vessels shown with FITC-LEA-labeled endothelium (green) and 4′,6-diamidino-2-phenyl-indole HCl-labeled nuclei (blue). (B) Summary of VE-cadherin fluorescence intensity changes in vascular segments. *P<0.05 for all, versus WT; †, versus Cbs+/− n=5 for all groups.
Figure 3
Figure 3
Expression caveolin-1 (Cav-1) and plasmalemmal vesicle-associated protein-1 (PV-1) in mouse brain vessels. (A) Examples of vessel images in samples obtained from wild type (WT), Cbs+/−, Cbs+/−, and matrix metalloproteinase-9 (Mmp9−/−) double knockout (Cbs+/−/Mmp9−/−), and MMP9 gene knockout (Mmp9−/−) mice; Expression of PV-1 (green) and Cav-1 (red) in cortical vessels shown with Lycopersicon esculentum agglutinin (LEA)-labeled endothelium (blue). (B–D) Summary of fluorescence intensity changes in brain cortical vessels defined by immunohistochemistry. Examples of western blot images of Cav-1 (E) and PV-1 (G) expressions in mouse brain cortical samples. Summary of ratios of integrated optical density (IOD) of Cav-1 (F) and PV-1 (H) bands from each group to those of the respective glyceraldehyde-3-phosphate dehydrogenase (GAPDH) bands. Note: Cell lysate (lysate) was used as a positive control for Cav-1. *P<0.05, versus WT; †, versus Cbs+/− n=4 for all groups.
Figure 4
Figure 4
Deposition of fibrinogen (Fg) and amyloid beta (Aβ) in mouse brain vessels. (A) Examples of vessel images in samples obtained from wild type (WT), Cbs+/−, Cbs+/−, and matrix metalloproteinase-9 (Mmp9−/−) double knockout (Cbs+/−/Mmp9−/−), and MMP9 gene knockout (Mmp9−/−) mice. Deposition of Fg (shown as red dots and indicated with red arrows) and Aβ (shown as green dots and indicated with green arrows), and their co-localization (shown in yellow and indicated with white arrows) in brain vessels was assessed by measuring fluorescence intensities of Fg (red) and Aβ (green) and number of spots with co-localized green and red colors after deconvolution of images. Summaries of Aβ (B) and Fg (C) depositions and Fg and Aβ co-localization (D) assessments. *P<0.05 for all, versus WT; †, versus Cbs+/− ‡, versus Cbs+/−/Mmp9−/− n=5 for all groups.
Figure 5
Figure 5
Deposition of amyloid beta (Aβ) on vascular collagen in mouse brain vessels. (A) Examples of vessel images in samples obtained from wild type (WT), Cbs+/−, Cbs+/−, and matrix metalloproteinase-9 (Mmp9−/−) double knockout (Cbs+/−/Mmp9−/−), and MMP9 gene knockout (Mmp9−/−) mice. Deposition of Aβ (green) and collagen (red) and their co-localization (shown in yellow and indicated by arrowheads) was assessed by measuring fluorescence intensities of Aβ (green) and collagen (red) and number of spots with co-localized green and red colors after deconvolution of images. (B) Summary of Aβ deposition assessment. (C) Summary of collagen expression assessment. (D) Summary of Fg and Aβ co-localization assessment. *P<0.05 for all, versus WT; †, versus Cbs+/− ‡, versus Cbs+/−/Mmp9−/− n=5 for all groups.
Figure 6
Figure 6
Hyperhomocysteinemia-induced loss of short-term memory in mice. Short-term memory of wild type (WT), Cbs+/−, Cbs+/− and matrix metalloproteinase-9 (Mmp9−/−) double knockout (Cbs+/−/Mmp9−/−), and MMP9 gene knockout (Mmp9−/−) mice was assessed by novel object recognition test. Lower discrimination ratio indicates impaired short-term memory. *P<0.05 for all, versus WT; †, versus Cbs+/− n=10 for all groups.
Figure 7
Figure 7
Possible mechanism of hyperhomocysteinemia (HHcy)-induced formation of fibrinogen–amyloid β (Fg–Aβ)-collagen complex formation. HHcy increases activity of matrix metalloproteinase-9 (MMP-9), which enhances cerebrovascular permeability mainly through paracellular transport pathway down regulating VE-cadherin. Increased vascular permeability leads to accumulation of Fg in subendothelial matrix and promotes its binding to Aβ and collagen resulting in formation of Fg–Aβ-collagen complex, which can be involved in short-term memory loss. Note: The schematic model depicting the possible mechanism of Fg–Aβ complex formation during HHcy presented above is not specific to brain vessels. However, brain is the place where Fg–Aβ complex formation and its possible association with neuronal cells have the most devastating effect.
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