This study demonstrates an elevation of CSF uPA in patients suffering from CAA pathology. In two separate cohort studies, CSF uPA levels, after adjustment for age, were significantly elevated in sCAA patients, when compared with respective controls. In contrast, no elevation of uPA levels was observed after adjustment for age in (a)symptomatic D-CAA patients, compared to their respective age-matched controls. Perivascular uPA in amyloid-affected cerebral vessels was overexpressed in rTg-DI rats and a sCAA patient. Moreover, uPA (mRNA) levels were increased in CSF from the rTg-DI CAA rodent model.
Earlier studies have demonstrated a decreased of uPA levels in plasma, but not CSF, in mild-cognitive impairment (MCI) patients with decreased Aβ42/Aβ40 ratios (indicative of amyloid pathology) compared to controls . Similarly, no differences in the CSF concentrations of plasminogen, tPA or plasminogen activator inhibitor-1 between AD patients and controls were discovered. Moreover, in this study zymography analysis in CSF revealed no difference in tPA activity between AD and controls, whereas uPA activity could not be detected in CSF of either AD or control cases . However, in one other study, increased levels of CSF tPA were observed in AD vs. patients with subjective cognitive impairment, but not in AD vs. MCI patients . In summary, these studies show that uPA as an influential factor in CAA is barely being researched and that uPA CSF levels are not increased in AD, whereas inconsistent results were obtained for tPA levels in AD. Despite the partial overlap in pathology of AD and CAA, elevated levels of uPA in CSF seem to be restricted to CAA. Future studies should reveal if uPA levels are increased in AD patients with severe CAA compared to AD cases without CAA .
Endothelial cells are a major source of vascular uPA expression, thereby suspected of major contributors to the activation of the plasminogen activation system, through the conversion of plasminogen into plasmin [30, 31]. Because of this role in plasmin production, uPA and other components of the plasminogen activation system, are often associated with tissue remodeling processes, like cellular migration and metastasis, hemostasis, fibrinolysis or angiogenesis . Earlier studies have shown elevated expression of both uPA and its receptor, uPAR, after stimulation of human smooth muscle cells with Aβ . Whereas the colocalization of uPA with Aβ in CAA patients and rTg-DI models we describe here has not been described before, tPA, the tissue-counterpart of uPA, has been found to colocalize with Aβ in the cerebral vasculature of Tg2576 mice, an AD mouse model . The co-localization of tPA and Aβ may be explained by the observations that both tPA and plasminogen bind to lysine-rich structures which are present both in fribrin and in Aβ .
Elevated cerebrovascular expression of uPA may stimulate the localized conversion of plasminogen to plasmin, which in a compensatory feedback-mechanism might directly decrease the vascular amyloid load through plasmin, a known Aβ-cleaver [20, 25, 33-35]. This proposed mechanism for the plasminogen activation system in cleaving (vascular) Aβ may also take place in senile plaques [36-43]. Whereas the uPA-induced elevated plasmin level may thus decrease vascular amyloid load, elevated plasmin levels may also negatively affect the integrity of the extracellular matrix (ECM) of affected tissues. This secondary effect could occur either directly, through plasmin-mediated focal degradation of matrix proteins, or indirectly, through the uPA- or plasmin-mediated activation of other proteolytic factors, such as the matrix metalloproteinases MMP-2, MMP-3, MMP-9, MMP-12 and MMP-13 [44-47]. Degradation of the ECM directly compromises the integrity of the vascular wall and potentially contributes to the development of cerebral (micro)haemorrhages associated with CAA.
Specific protein biomarkers in CSF may discriminate between CAA patients, AD patients and/or control subjects: e.g. decreased CSF Aβ40 discriminates sCAA from controls [12, 14, 48]. These previous studies also revealed decreased CSF Aβ42 levels in sCAA patients in comparison with controls. Also, in D-CAA decreased CSF Aβ40 and Aβ42 levels were found in asymptomatic and symptomatic mutation carriers in comparison with age-matched controls . However, CSF Aβ42 levels were similarly decreased in AD patients as well, limiting the specificity of CSF Aβ42 as a biomarker for CAA. Other protein biomarkers have been associated with sCAA, but again, with limited specificity (e.g. CSF Apolipoprotein D) .
We observed no significant differences in CSF uPA levels between symptomatic D-CAA patients (either unadjusted or when adjusted for age of subjects) and asymptomatic D-CAA patients (borderline insignificant, after adjustment for age of subjects). We did however observe a difference in unadjusted CSF uPA levels between asymptomatic D-CAA patients and controls. This could be induced by age-effects (as the difference is not present in our age-adjusted model), but could also have a physiological cause. We can only speculate on the origins of this difference, but it is not unlikely that progessive deposition of Aβ induces an elevation of uPA expression and secretion in sCAA and asymptomatic D-CAA patients. The loss of uPA-producing endothelial cells following cerebral vessel rupture, may lead to a normalization of uPA expression in symptomatic D-CAA patients relative to asymptomatic D-CAA patients. Longitudinal studies on changes in CSF uPA levels in sCAA and D-CAA patients may yield more insight into these possible sequence of events .
Several limitations apply to our study. First, the number of rats included in the CSF study was relatively small. However, the homogeneity of this particular transgenic rat population reduces the influence of confounding factors. Second, despite the observation that our clinical cohorts were of relatively moderate sizes, these sizes are comparable to those in previously peer-reviewed CAA studies, which have been proven capable of producing reliable results [14, 50]. Third, it would have been interesting to also include patients suffering from AD (with vs. without evidence of CAA) and hypertensive vasculopathy as a different cause of haemorrhages in this study to address the specificity of our findings. Unfortunately these samples were not available to us, but including them in future studies could offer novel opportunities for research about the specificity of this biomarker.
Strengths of our study include the use of a unique animal model of CAA. This animal model not only recapitulates many of the characteristics of human CAA, but also allows for relatively easy collection of CSF for biomarker studies. Furthermore, the cohorts of all types of CAA patients (sporadic, asymptomatic D-CAA and symptomatic D-CAA) and controls (well-phenotyped using MRI, CSF and clinical data) are unique and a valuable source for studies of novel biomarkers of both sporadic, as well as hereditary CAA. Finally, the similarities between the animal and human findings strengthen the translational capacity of the rat model to study the pathogenesis of CAA and vice versa.
In conclusion, our findings show a multifaceted relationship between uPA and CAA pathophysiology, in both the rTg-DI CAA model and in CAA patients. Increased cerebrovascular uPA expression was observed in both rat and human CAA tissue; elevated CSF uPA levels were found in both rTg-DI CAA model rats and sCAA patients (after adjustments for age). Symptomatic D-CAA patients did not show robust differences when compared to control groups, but CSF uPA levels trended towards an elevation in asymptomatic D-CAA patients vs. controls. The association of uPA with CAA provides new insights in pathophysiological processes in both sporadic and hereditary CAA and could serve as a vascular biomarker for CAA in conjunction with other CAA-specific biomarkers.