Background Current diagnostic criteria for cerebral amyloid angiopathy (CAA) are predominantly based on radiological identification of evidence of CAA (micro‐ or macrobleeds). These criteria present only end‐stage manifestations of the disease, however. The development of transgenic rat models for CAA, carrying the E693Q/D694N mutations in APP (rTg‐DI rats), has the potential of discovery of novel biomarkers for CAA. Shotgun proteomics analysis of rTg‐DI tissue, compared to wild‐type rats has yielded elevated levels of urokinase plasminogen activator (uPA) in CAA. uPA is a serine protease active in the conversion of plasminogen to plasmin, a fibrinolytic factor. We investigated the potential of uPA in a pilot biomarker discovery study for the diagnosis of CAA by analyzing uPA concentrations in cerebrospinal fluid (CSF) of patients with CAA compared to control patients. Method CSF was obtained from patients with possible or probable CAA (according to the current diagnostic imaging tool, the Boston Criteria) (n=28), and control subjects (n=42). uPA levels in CSF were determined using a uPA Quantikine ELISA (R&D Systems, Minneapolis, USA). Total protein levels were assayed using a Pierce® BCA Protein Assay (Thermo Fisher, Waltham, USA). Associations of uPA with known concentrations of other neurological markers, including aβ peptides and tau proteins were also analysed. Result The concentration of uPA was significantly increased in the CSF of CAA patients compared to controls (303 ± 23.2 vs. 227 ± 12.5 pg/mL; p=0.001). CSF uPA levels (very) weakly correlated with age at lumbar puncture (rSP=0.33, p=0.005) and total protein content (rSP=0.24, p=0.042). uPA levels in controls were correlated in a weak‐to‐moderate extent with Aβ‐38, 40, 42, and total‐ and phosphorylated tau protein (rSP=0.46, 0.63, 0.47, 0.53 and 0.41 respectively, p<0.02), whereas this correlation was practically absent in the CAA group. Conclusion Comparison of CSF levels of uPA shows a significant elevation in CAA patients compared against controls. CAA patients show lower levels of correlation of uPA with circulating aβ peptides and tau protein variants as compared to controls. This reinforces the potential of uPA as an effective biomarker in the diagnosis of CAA patients and incentivizes further research into this biomarker.

Background To evaluate the potential of cerebrospinal fluid (CSF) levels of matrix metalloproteinases and tissue-type inhibitors (MMP; TIMP), and ratios of MMPs to TIMPs, to function as biomarkers for sporadic or hereditary cerebral amyloid angiopathy (CAA). Methods CSF concentrations of the matrix metalloproteinases MMP-2, MMP-9 and MMP-14, as well as the tissue inhibitors of metalloproteinases TIMP-1, TIMP-2 and TIMP-3, were determined using immunoassays. These assays were applied to two, independent study groups of sporadic CAA (sCAA) (n = 28/43) and control subjects (n = 40/40), as well as to groups of pre-symptomatic (n = 11) and symptomatic hereditary Dutch-CAA (D-CAA) patients (n = 12), and age-matched controls (n = 22/28, respectively). Results In the sCAA/control cohorts, inconsistent differences were found for individual MMPs and TIMPs, but MMP-2/TIMP-2 (discovery/validation: p = 0.004; p = 0.02) and MMP-14/TIMP-2 ratios (discovery/validation: p < 0.001; p = 0.04) were consistently decreased in sCAA, compared to controls. Moreover, MMP-14 was decreased in symptomatic D-CAA (p = 0.03), compared to controls. The MMP-14/TIMP-1 (p = 0.03) and MMP-14/TIMP-2 (p = 0.04) ratios were decreased in symptomatic D-CAA compared to controls and also compared to pre-symptomatic D-CAA (p = 0.004; p = 0.005, respectively). Conclusion CSF MMP-2/TIMP-2 and MMP-14/TIMP-2 were consistently decreased in sCAA, compared to controls. Additionally, MMP-14/TIMP-2 levels were also decreased in symptomatic D-CAA, compared to both pre-symptomatic D-CAA and controls, and can therefore be considered a biomarker for sporadic and late-stage hereditary forms of CAA.

Background: Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are involved in extracellular matrix (ECM) modelling and regulation of basement membrane integrity. A chronic disbalance in the expression and activity of these proteinases and their inhibitors in favor of MMPs can lead to excessive degradation of the ECM, dysfunction of the blood-brain barrier (BBB) and subsequent (micro)bleeds. Cerebral amyloid angiopathy (CAA) is characterized by accumulation of the amyloid-β (Aβ) protein in cerebral vessel walls and decreased BBB integrity, and is a major cause of intracerebral hemorrhage and cognitive decline. Currently, diagnosis of CAA is based on neuroimaging, but there is a need for alternative methods as these neuroimaging methods are not optimal for detection of CAA during life at an early stage. Previously, Aβ40 levels in the cerebrospinal fluid (CSF) have been suggested as promising biomarker for CAA. We hypothesize that a disbalance in MMP/TIMP expression is associated with CAA and is reflected in the CSF, and may besides Aβ40, be a potential biomarker candidate for detection of CAA at an early stage. In this study, we aimed to quantify levels of MMPs, TIMPs and Aβ proteins in the CSF to gain more insight in the pathophysiology of CAA and to establish their potential biomarker value for CAA. Method:We used immunoassays to quantify MMP-14, TIMP-1 and Aβ40 levels in CSF in a group of controls (n=40) and CAA patients (n=28). Result:The MMP-14/TIMP-1 ratio was decreased in CAA (0.016 ± 0.005) vs. controls (0.022 ± 0.008; p=0.0007), which was a stronger difference than observed for either MMP-14 (not significant: p=0.53) or TIMP-1 (p=0.0027) alone, or for Aβ40 (p=0.038).The difference of the MMP-14/TIMP-1 ratio was robust and remained after correction for age and total protein levels. Conclusion:Our results suggest that a disbalance in the expression of MMP-14 and TIMP-1 in CSF is associated with CAA, and may be a promising biomarker both to identify CAA, with a higher diagnostic value than CSF Aβ40, as well as to obtain more insight into the pathophysiological processes in CAA.

Background Diagnosis of the highly prevalent disease cerebral amyloid angiopathy (CAA) is mainly based on radiological identification of cerebral micro‐ or macrobleeds (according to the diagnostically acclaimed Boston Criteria) in the end‐stage of disease. Body fluid biomarkers (e.g. in cerebrospinal fluid) could potentially act as alternatives and allow for earlier identification of disease in a minimally invasive manner. We have explored the involvement of potential CAA‐biomarker urokinase plasminogen activator (uPA) in CAA development and progression in human patients and rodent disease models (rTg‐DI). Additionally, we analyzed uPA cerebrospinal fluid (CSF) levels in rTg‐DI models and human CAA patients versus controls to assess diagnostic accuracy of uPA as a biomarker for CAA. Method PLAU gene expression and uPA localization were studied in cerebrovascular tissue of a rTg‐DI models and wild‐type rats, alongside a human sporadic CAA patient and a control subject, using rt‐qPCR and immunohistochemistry respectively. Additionally, CSF Aβ40 and uPA levels were determined in rTg‐DI rats and in human patients with possible or probable CAA (according to the Boston Criteria; n=28), and control subjects (n=40), using ELISA. Result Immunohistochemistry showed strong overexpression of uPA in brain tissue of rTg‐DI models and human CAA patients. Additionally, uPA showed a strong colocalization with Aβ peptides, restricted to the vasculature of rTg‐DI rats and human CAA patients. Expression and localization of both uPA and Aβ peptides were negligible in controls. Additionally, CAA rats displayed significant overexpression of the PLAU gene in brain vasculature as well as significant elevation of uPA in their CSF. In humans, CSF Aβ40 levels were reduced in CAA patients (7.66 ± 3.36 ng/mL) compared to controls (9.58 ± 3.88 ng/mL, p=0.04). CSF uPA levels were higher in CAA patients (median (IQR): 92.0 (76.1 ‐ 109.0) pg/mL compared to human controls (68.5 (56.6 ‐ 78.4) pg/mL; p<0.001). CSF Aβ40 and uPA combined yielded the highest AUC (0.87) to distinguish CAA from controls. Conclusion uPA was overexpressed in rTg‐DI rats and its levels were significantly elevated in both rodent and human CAA. CSF Aβ40 and uPA serve as excellent biomarkers to discriminate CAA from controls, especially when combined.

Aims: The aim of this work is to study the association of urokinase plasminogen activator (uPA) with development and progression of cerebral amyloid angiopathy (CAA). Materials and methods:We studied the expression of uPA mRNA by quantitative polymerase chain reaction (qPCR) and co-localisation of uPA with amyloid-β (Aβ) using immunohistochemistry in the cerebral vasculature of rTg-DI rats compared with wild-type (WT) rats and in a sporadic CAA (sCAA) patient and control subject using immunohistochemistry. Cerebrospinal fluid (CSF) uPA levels were measured in rTg-DI and WT rats and in two separate cohorts of sCAA and Dutch-type hereditary CAA (D-CAA) patients and controls, using enzyme-linked immunosorbent assays (ELISA).Results: The presence of uPA was clearly detected in the cerebral vasculature of rTg-DI rats and an sCAA patient but not in WT rats or a non-CAA human control. uPA expression was highly co-localised with microvascular Aβ deposits. In rTg-DI rats, uPA mRNA expression was highly elevated at 3 months of age (coinciding with the emergence of microvascular Aβ deposition) and sustained up to 12 months of age (with severe microvascular CAA deposition) compared with WT rats. CSF uPA levels were elevated in rTg-DI rats compared with WT rats (p = 0.03), and in sCAA patients compared with controls (after adjustment for age of subjects, p = 0.05 and p = 0.03). No differences in CSF uPA levels were found between asymptomatic and symptomatic D-CAA patients and their respective controls (after age-adjustment, p = 0.09 and p = 0.44). Increased cerebrovascular expression of uPA in CAA correlates with increased quantities of CSF uPA in rTg-DI rats and human CAA patients, suggesting that uPA could serve as a biomarker for CAA.

Objective: To study the association of urokinase plasminogen activator (uPA) with development and progression of cerebral amyloid angiopathy (CAA) in a rat model for CAA type-I (rTg-DI) and in patients with sporadic (sCAA) and Dutch-type hereditary CAA (D-CAA). Additionally, to investigate the potential of uPA to serve as a diagnostic biomarker of CAA in cerebrospinal fluid (CSF) from patients with sCAA and D-CAA.Methods: We studied the expression of uPA mRNA by qPCR and co-localization of uPA with amyloid-β (Aβ) using immunohistochemistry in the cerebral vasculature of rTg-DI rats compared to wild type (WT) rats and in an sCAA patient and control subject using immunohistochemistry. CSF levels of uPA were measured in rTg-DI and WT rats and in two separate cohorts of sCAA and D-CAA patients and controls, using enzyme-linked immunosorbent assays (ELISA).Results: The presence of uPA was clearly detected in the cerebral vasculature of rTg-DI rats and an sCAA patient, but not in WT rats or a non-CAA human control. uPA expression was highly co-localized with microvascular Aβ deposits. In rTg-DI rats, uPA mRNA expression was highly elevated at 3 months of age (coinciding with the emergence of microvascular CAA deposition) and sustained up to 12 months of age (with severe microvascular CAA deposition) compared to WT rats. CSF uPA levels were elevated in rTg-DI rats compared to WT rats (p=0.03), and in two separate, independent groups of sCAA patients compared to controls (after adjustment for age of subjects, p=0.05 and p=0.03). No differences in CSF uPA levels were found between asymptomatic and symptomatic D-CAA patients and their respective controls (after age-adjustment, p=0.09 and p=0.44).Conclusion: uPA expression appears linked to the onset of CAA in rTg-DI rats. Increased cerebrovascular expression of uPA in CAA correlates with increased quantities of CSF uPA in rTg-DI rats and human CAA patients, suggesting that uPA could serve as a biomarker for CAA. No differential expression levels of uPA in CSF of (a)symptomatic D-CAA patients were discovered. These studies also further support the use of rTg-DI rat models as a useful preclinical model for the study of human CAA type-I.