Combinatorial Treatment Using Umbilical Cord Perivascular Cells and Aβ Clearance Rescues Vascular Function Following Transient Hypertension in a Rat Model of Alzheimer Disease

Abstract

Transient hypertension is a risk factor for Alzheimer disease (AD), but the effects of this interaction on brain vasculature are understudied. Addressing vascular pathology is a promising avenue to potentiate the efficacy of treatments for AD. We used arterial spin labeling magnetic resonance imaging to longitudinally assess brain vascular function and immunohistopathology to examine cerebrovascular remodeling and amyloid load. Hypertension was induced for 1 month by administration of l-N^G-nitroarginine-methyl-ester in TgF344-AD rats at the prodromal stage. Following hypertension, nontransgenic rats showed transient cerebrovascular changes, whereas TgF344-AD animals exhibited sustained alterations in cerebrovascular function. Human umbilical cord perivascular cells in combination with scyllo-inositol, an inhibitor of Aβ oligomerization, resulted in normalization of hippocampal vascular function and remodeling, in contrast to either treatment alone. Prodromal stage hypertension exacerbates latter AD pathology, and the combination of human umbilical cord perivascular cells with amyloid clearance promotes cerebrovascular functional recovery.

Keywords: Alzheimer disease; hypertension; inositol; risk factor; umbilical cord.

Figure 1.

Experimental approach. A, Experimental timeline; (B) baseline hemodynamics. Resting hippocampal perfusion (left: 74.2±3.4 mL/[min·100 mL] tissue for the nontransgenic [non-Tg] vs 71.1±1.9 mL/[min·100 mL] tissue for the TgF344-AD, P=0.5) and cerebrovascular responsivity to hypercapnia (right: 24.8±4.5% for the non-Tg vs 29.3±2.9% tissue for the TgF344-AD, P=0.4) were indistinguishable between the 2 genotypes at baseline. C, N(G)-Nitro-l-arginine methyl ester (l-NAME) effect on blood pressure (BP) and PAI1 (plasminogen activator inhibitor-1). C, left, Blood pressure rose from baseline level of 137.3±3.9 to 152.7±4.5 mm Hg at the end of the l-NAME administration (P=0.02); the latter time point coincided with post–l-NAME time point of magnetic resonance imaging (MRI) experiments. One month after termination of l-NAME treatment (ie, at recovery time point) BP normalized to baseline levels (129.3±2.5 mm Hg, P=0.3 when compared with baseline, P=0.0002 when compared with post–l-NAME, 1-way ANOVA with post hoc Tukey honestly significant difference test). Right, PAI1 concentration rose from baseline level of 72.4±8.3–105.8±13.2 pg/mL at the end of the l-NAME administration (P=0.041); the latter time point coincided with post–l-NAME time point of MRI experiments. One month after termination of l-NAME treatment (ie, at recovery time point) PAI1 concentration normalized to baseline levels (81.3±12.4 pg/mL, P=0.56 when compared with baseline, P=0.5 when compared with post–l-NAME). CASL indicates continuous arterial spin labeling; CBF, cerebral blood flow; and HUCPVC, human umbilical cord perivascular cell.

Figure 2.

Cerebral hemodynamics in nontransgenic (non-Tg) animals. Non-Tg rats show a drop in the resting perfusion from 76.7±0.96 mL/(min·100 mL) tissue at baseline to 45.8±4.5 mL/(min·100 mL) post–l-NAME, which then recovers to 57.6±11.5 mL/(min·100 mL) at the end point (A, P=0.00015 and P=0.53 respectively, Wilcoxon rank-sum test with Bonferroni adjustment). Non-Tg rats’ responsivity to hypercapnia increases from 37.8±11.1% at baseline to 67.9±10.8% post–l-NAME and then recovers to 71.0±21.9% at the end point (B, P=0.0066 and P=0.21, respectively, Wilcoxon rank-sum test with Bonferroni adjustment). Sham l-NAME non-Tg animals showed resting perfusion and responsivity to hypercapnia similar to Sham administered non-Tg animals at baseline (resting perfusion: 93.1±17 vs 76.7±0.96 mL/min/100 mL tissue, P=0.12; hypercapnia challenges: 57.7±23.1 vs 37.8±11.1%, P=0.65). CBF indicates cerebral blood flow.

Figure 3.

Human umbilical cord perivascular cell markers. Flow cytometry experiments targeting cell surface mesenchymal stromal cell markers show expression of CD90-PE, CD34-APC, CD44-FITC, CD105-FITC, CD73-PEVio770, and negative or low expression of nonmesenchymal markers CD45-PE, HLA-DPQR-APC, CD31-VioBlue. APC indicates activated protein C; FITC, fluorescein isothiocyanate; and PE, phycoerythrin.

Figure 4.

Resting perfusion in TgF344-AD rats. A, Representative continuous arterial spin labeling (CASL) cerebral blood flow (CBF) maps showing a drop in resting perfusion in the hippocampi (white arrows) of TgF344-AD rats from baseline, to post–l-NAME and the perfusion contrast between vehicle administration vs scyllo-inositol (SI) + human umbilical cord perivascular cell (HUCPVC) treatment at end point. B, Population analysis reveals a significant drop in resting perfusion from 73.8±1.6 at baseline to 48.3±3.8 mL/(min·100 mL) tissue post–l-NAME (P=8.52×10⁻⁶). Resting perfusion in vehicle administered animals at end point was still significantly reduced when compared with that at baseline (46.8±1.1 mL[min·100 mL] tissue, P=0.000235). SI-only and HUCPVC-only treated cohorts were indistinguishable from vehicle administered group (P=0.8 and P=0.14, respectively), whereas animals treated with a combination of SI and HUCPVC showed improvement in resting perfusion (63.9±1.9 mL/[min·100 mL] tissue, P=0.06 when compared with the vehicle-administered group). At end point, the TgF344-AD Sham l-NAME group showed higher resting perfusion than did the l-NAME and vehicle administered (77.7±7.7 vs 47±1 mL/[min·100 mL] tissue, P=0.000476).

Figure 5.

Cerebrovascular reactivity in the TgF344-AD rats. A, Representative experiments showing increased responsivity to hypercapnia in the hippocampus (white arrows) of TgF344-AD rats from baseline to post–l-NAME and at end point in a vehicle-administered animal but with relative resolution of this hyperreactivity in the scyllo-inositol (SI) + human umbilical cord perivascular cell (HUCPVC) treated rat. B, Averaged (black line and SD in gray) time courses of the responses over 4 hypercapnic challenges are shown at baseline, post–l-NAME and at end point for vehicle administered and SI+HUCPVC treated subject. Population analysis (C) reveals significantly increased (P=0.000105) responsivity to CO₂ challenges from baseline (29.4±3.9%) to post–l-NAME (92.7±12.4%). The responsivity to hypercapnia remained increased at the end point in vehicle-administered rats when compared with baseline (106.1±5.5%, n=7, P=0.000381). When compared with vehicle-administered animals, SI-only treated rats did not show significant reduction in the cerebrovascular responses to hypercapnia (111.5±27.7%, P=0.87); similarly, HUCPVC-only treated rats showed no significant reduction in the cerebral blood flow (CBF) responses to hypercapnia (88.9±25.9%, P=0.42), whereas rats that received a combined treatment of SI+HUCPVC showed a pronounced reduction in their CBF responses to hypercapnia (31.2±18.4%, P=0.01). At end point, the TgF344-AD Sham l-NAME group showed reduced responses to hypercapnia than did the l-NAME and vehicle administered (51.6±16.2% vs 106.1±5.5%, P=0.006758).

Figure 6.

Cerebrovascular pathology and Aβ plaques in the TgF344-AD rats. Hippocampal arterioles stained for elastin (left) and collagen IV (middle), and overlay (right) in a representative vehicle-treated TgF344-AD rat (A), in an scyllo-inositol (SI) + human umbilical cord perivascular cell (HUCPVC) treated TgF344-AD rat (B) and in a TgF344-AD Sham l-NAME (C); scale bar 25 m. Population analysis (D) showed that combined treatment of SI+HUCPVC increased the elastin to collagen IV ratio. E, Representative 6F3D staining for Aβ plaques in the hippocampus of a vehicle-administered TgF344-AD rat (top left), SI-only treated TgF344-AD rat (top right), HUCPVC-only treated TgF344-AD rat (middle left), SI+HUCPVC treated TgF344-AD rat (middle right), and a TgF344-AD Sham l-NAME rat (bottom left). Scale bar: 0.8 mm. Population analysis (E) revealed a lower Aβ plaque load in SI-only and SI+HUCPVC treated TgF344-AD rats when compared with that of vehicle-administered and HUCPVC-only treated animals.