Plasma-type Gelsolin is Decreased in Human Blood and Cerebrospinal Fluid following Subarachnoid Hemorrhage

Abstract

Background

Subarachnoid hemorrhage (SAH) pathophysiology involves neurovascular proteolysis and inflammation. How these two phenomena are related remains unclear. We hypothesize that matrix metalloproteinases (MMPs) mediate the depletion of anti-inflammatory plasma-type-gelsolin (pGSN).

Methods

We enrolled 42 consecutive SAH subjects and sampled CSF and blood on post-SAH days 2–3, 4–5, 6–7, and 10–14. Controls subjects were 20 consecutive non-SAH hydrocephalus patients with lumbar-drains. LISA, western blotting and zymography were used to quantify pGSN and MMP-9.

Results

In CSF, pGSN was lower in SAH compared with control subjects on post-SAH days 2–3 (p =0.0007), 4–5 (p=0.041), and 10–14 (p=0.007). In blood, pGSN decreased over time (p=0.001), and was lower in SAH compared to control subjects on post-SAH days 4–5 (p=0.037), 6–7 (p=0.006), and 10–14 (p=0.006). Western blots demonstrated that SAH CSF had novel bands at 52 and 46 kD, representing cleaved pGSN fragments. Gelatin zymography showed that CSF MMP-9 was elevated in SAH compared with controls. Higher CSF MMP-9 correlated with lower CSF pGSN on post-SAH day 7 (r= −0.38; p= 0.05).

Conclusion

SAH is associated with decreased CSF and blood pGSN and elevated CSF MMP-9. Novel cleaved pGSN fragments are present in CSF of SAH subjects, consistent with pGSN cleavage by MMPs. Since pGSN is known to inhibit inflammatory mediators, these findings suggest that MMPs may reduce pGSN and exacerbate inflammation after SAH. Further studies are warranted to investigate the mechanisms underlying MMP-pGSN signaling in SAH.

INTRODUCTION

Cerebrovascular inflammation following SAH may mediate vasospasm and poor outcomes.¹ Plasma-type-gelsolin (pGSN) scavenges circulating actin and inhibits peptide and lipid mediators of inflammation.² Total gelsolin depletion increases neuronal death following focal cerebral ischemia in mice.^{3, 4} Clinically, blood pGSN is decreased in critical illness,⁵ and lower blood pGSN predicts trauma and critical illness mortality.⁶ CSF pGSN is reduced in neuroinflammatory diseases such as multiple sclerosis.⁷ Matrix metalloproteinases (MMPs),⁸ which are elevated in CSF of SAH patients,⁹ cleave pGSN. We hypothesize that blood and CSF pGSN depletion occurs in critically ill SAH patients, and that this may in part be caused by elevated MMPs.

METHODS

Consecutive consenting SAH patients (n=42) and hydrocephalus patients without brain injury (n=20) were recruited as case and controls. SAH subjects are included if they are 18 and older, within 96 hours of spontaneous SAH and have an external ventricular drain (EVD) placed for clinical indication. Blood and CSF were collected on post-SAH days 2–3, 4–5, 6–7, and 10–14. Patients with secondary SAH, pregnancy, end-stage renal or hepatic disease, intracranial malignancies, or infectious meningitis were excluded. CSF was obtained from existing EVD or lumbar drains. All subjects were consented following IRB-approved protocols.

Blood and CSF samples were immediately centrifuged (3900 RPM, 15 minutes), aliquotted, and stored at −80C. pGSN was quantified via ELISA (KSB BioTechnology). Western blot were performed using antibodies specific to carboxyl and amino terminals of human pGSN. MMP-9 was assessed by ELISA (R&D systems) and gelatin zymography.

Continuous variables between independent groups were compared using student’s t-test or Wilcoxan Rank Sum test depending on data normality. Repeated measurements were compared using longitudinal regression. Correlations were performed using Spearman correlation (SAS 9.2).

RESULTS

Study subject demographics are summarized in Table 1. Blood pGSN levels in SAH (2236–3737 u/L; SD = 1395–2459 u/L) were significantly lower than controls (5792 u/L; SD = 4535 u/L) (Figure 1a). Furthermore, blood pGSN levels on post-SAH days 5–14 were significantly lower than initial levels on days 2–3 (p=0.002), and blood pGSN levels decreased over time (p=0.001).

Table 1.

Patient Demographics

		SAH (n = 42 )	Control (n= 20 )
Mean Age		53.6 (SD 16.4)	72.7 (SD 11.0)
Gender (% Female)		24 (57%)	1 (5%)
HH Grade			n/a
1		5 (12%)
2		14 (33%)
3		10 (24%)
4		8 (19%)
5		5 (12%)
Fisher Grade			n/a
1		2 (5%)
2		6 (14%)
3		32 (76%)
4		2 (5%)
Angiographic Vasospasm			n/a
Yes		22 (52%)
no		20 (48%)
Aneurysm Location			n/a
Anterior Communicating Artery		20 (48%)
Posterior Communicating Artery/Internal Carotid Artery		11 (26%)
Angio-negative SAH		4 (10%)
Posterior Inferior Cerebellar Artery		3 (7%)
Anterior Cerebral Artery		2 (5%)
Middle Cerebral Artery		1 (2%)
Basilar Artery		1 (2%)
3 Month Clinical Outcome by Modified Rankin Score (mRS)			n/a
mRS ≤ 2		28 (68%)
mRS > 2		13 (32%)

Figure 1.

(a) Blood pGSN over time in SAH and in normal controls. (b) CSF pGSN overtime in SAH and in normal controls.

pGSN was detectable in CSF from both SAH and controls, with CSF pGSN approximately 10-fold lower than blood pGSN. CSF pGSN in SAH (92–125 u/L) were significantly lower than controls (148 u/L) on all post-SAH days except for post-SAH days 6–7 (Figure 1b). CSF pGSN levels were not associated with angiographic vasospasm.

Western blot of SAH CSF showed cleaved pGSN bands at 52 and 46 KD that were not seen in controls (Figure 2a). The molecular weights of these cleaved bands are similar to those from in vitro MMP digestion of pGSN.⁸ Gel zymography of CSF from the same subjects showed elevation of MMP-9 but not MMP-2 after SAH (Figure 2b). Higher CSF MMP-9 correlated with lower CSF pGSN on post-SAH day 7 (r= −0.38; p= 0.05) (Figure 3).

Figure 2.

(a) Cleaved pGSN fragments are present in CSF of SAH but not controls. (b) CSF MMP-9 in SAH is elevated compared with controls. Three representative cases from controls (C1-C3) and SAH (S1-S3) are shown.

Figure 3.

CSF MMP9 and pGSN correlation in SAH.

DISCUSSION

In this study, we explored blood and CSF pGSN as potential biomarkers linking neurovascular proteolysis with inflammation after SAH. We report for the first time that blood pGSN is reduced in SAH, as in sepsis and trauma. We also found CSF pGSN to be reduced in SAH compared with controls. Interestingly, novel CSF pGSN fragments were detected in SAH but not in control subjects, suggesting possible disease-specific pGSN proteolytic cleavage in CSF space. Elevated MMP-9 after SAH and the inverse correlation between MMP-9 and pGSN levels in CSF suggests that MMP-9 may be one of the neurovascular proteases responsible for pGSN cleavage.

Taken together, our findings may provide a mechanistic link between neurovascular proteolysis and inflammation after SAH. However, this proof-of-concept study has several caveats. First, our small sample size limits power to detect small subgroup differences. Because critical illness in general may lower pGSN, we cannot rule out the possibility that changes in blood pGSN here may reflect nonspecific reaction to systemic illness in SAH. Second, our control subjects have no acute brain injuries, but we cannot unequivocally exclude other brain pathology (e.g. Alzheimers) that might influence CSF pGSN. In addition, control CSF were sampled via lumbar drain while SAH CSF were sampled from an EVD. Recent CSF proteomic studies suggest that there is no clear ventriculo-lumbar gradient in low molecular weight CSF proteins,¹⁰ but we cannot exclude the possibility that different CSF sampling routes may affect our results. Third, we only explored MMP-2 and MMP-9. Our finding of elevated MMP-9 is consistent with experimental data, where MMP-9 contributes to SAH pathophysiology.¹¹ However, other MMPs and their inhibitors may also play a role. Finally, although this is the first report of pGSN cleavage in CSF, future effort is required to identify and quantify these novel pGSN fragments in SAH patients.

SAH is a challenging problem involving parallel mechanisms of neurovascular proteolysis and inflammation.¹² Our study points to a novel link between elevations in MMP-9 and reduction of the putative anti-inflammatory mediator pGSN. Blood and CSF pGSN and MMP-9 may be potential biomarkers and therapeutic targets for SAH. Larger studies with simultaneous quantification of pGSN, pGSN fragments, and other MMPs are warranted.