Fibrinogen-Induced Increased Pial Venular Permeability in Mice

Abstract

Elevated blood level of Fibrinogen (Fg) is commonly associated with vascular dysfunction. We tested the hypothesis that at pathologically high levels, Fg increases cerebrovascular permeability by activating matrix metalloproteinases (MMPs). Fibrinogen (4 mg/mL blood concentration) or equal volume of phosphate-buffered saline (PBS) was infused into male wild-type (WT; C57BL/6J) or MMP-9 gene knockout (MMP9À/À) mice. Pial venular leakage of fluorescein isothiocyanate-bovine serum albumin to Fg or PBS alone and to topically applied histamine (10 À5 mol/L) were assessed. Intravital fluorescence microscopy and image analysis were used to assess cerebrovascular protein leakage. Pial venular macromolecular leakage increased more after Fg infusion than after infusion of PBS in both (WT and MMP9À/À) mice but was more pronounced in WT compared with MMP9À/À mice. Expression of vascular endothelial cadherin (VE-cadherin) was less and plasmalemmal vesicle-associated protein-1 (PV-1) was greater in Fg-infused than in PBS-infused both mice groups. However, in MMP9À/À mice, VE-cadherin expression was greater and PV-1 expression was less than in WT mice. These data indicate that at higher levels, Fg compromises microvascular integrity through activation of MMP-9 and downregulation of VE-cadherin and upregulation of PV-1. Our results suggest that elevated blood level of Fg could have a significant role in cerebrovascular dysfunction and remodeling.

Ablation of MMP9 Gene Ameliorates Paracellular Permeability and Fibrinogen–Amyloid Beta Complex Formation during Hyperhomocysteinemia

“…For genotyping of Cbs þ / À /Mmp9 À / À mice, DNA was extracted from the tail tip of the mice and was amplified by polymerase chain reaction using specific primer sequences according to the protocol provided by the Jackson Laboratory and presented previously. 29 The primer sequences to identify Mmp9 À / À were forward: 5-CTG AAT GAA CTG CAG GAC GA-3 0 ; reverse: 5 0 -ATA CTT TCT CGG CAG GAG CA 3 0 for MMP9 mutations and for WT were forward: 5 0 -GTG GGA CCA TCA TAA CAT CAC A-3 0 ; reverse: 5 0 -CTC GCG GCA AGT CTT CAG AGT A-3 0 . The primer sequences to identify Cbs gene mutation were as follows: reverse 5 0 -CGT GCA ATC CAT CTT GTT CA-3 0 for Cbs mutant, reverse 5 0 -AGC CAA CTT AGC CCT TAC CC-3 0 for WT, and forward 5 0 -GAT TGC TTG CCT CCC TAC TG-3 0 for common.…”

“…Brain pial microcirculation was prepared for observations as previously described. 17,[29][30][31] Briefly, a mouse was placed on a stereotaxic apparatus (World Precision Instruments, Sarasota, FL, USA). The scalp and connective tissues were removed over the parietal cranial bone above the left hemisphere.…”

“…29 Animals killed with an anesthetic overdose were immediately infused with phosphate-buffered saline through the left ventricle for exsanguination. After the cranium was opened, the brain was gently dissected and removed for fresh tissue processing.…”

Sphingolipids affect fibrinogen-induced caveolar transcytosis and cerebrovascular permeability

“…Cranial windows were prepared and changes in pial venular permeability were observed as described previously (41). After surgical preparation, following a 1-h equilibration period, a mixture of 100 l of FITC-BSA (300 g/ml) and 20 l of myriocin or PBS in the respective control group was infused through the carotid artery cannula and allowed to circulate for 10 min (34,41). Brain pial circulation was observed with a microscope (model BXG61WI, Olympus, Tokyo, Japan) equipped with a ϫ10/0.40 (UPlanSApo, Olympus) objective.…”

“…Mean arterial blood pressure and heart rate were monitored through a carotid artery cannula connected to a transducer and a blood pressure analyzer (CyQ 103/302, Cybersense, Lexington, KY). Cranial windows were prepared and changes in pial venular permeability were observed as described previously (41). After surgical preparation, following a 1-h equilibration period, a mixture of 100 l of FITC-BSA (300 g/ml) and 20 l of myriocin or PBS in the respective control group was infused through the carotid artery cannula and allowed to circulate for 10 min (34,41).…”