Advanced glycation end products accumulate in vascular smooth muscle and modify vascular but not ventricular properties in elderly hypertensive canines

Abstract

Background: Advanced glycation end products (AGEs) are believed to increase left ventricular (LV) and vascular stiffness, in part via cross-linking proteins. We determined whether and where AGEs were increased in elderly hypertensive nondiabetic dogs and whether an AGE cross-link breaker (ALT-711) improved vascular or ventricular function.

Methods and results: Elderly dogs with experimental hypertension (old hypertensives [OH]) were randomized to receive ALT-711 (OH+ALT group; n=11; 1 mg/kg PO) or not (OH group; n=11) for 8 weeks. Conscious blood pressure measurements (weekly), echocardiography (week 8), and anesthetized study (week 8) with LV pressure-volume analysis and aortic pressure-dimension and pressure-flow assessment over a range of preloads and afterloads were performed. In LV and aorta from OH, OH+ALT, and young normal dogs, AGE content (immunohistochemistry and Western analysis for N(epsilon)-(carboxymethyl)lysine [CML]) was assessed. Aortic CML content was markedly increased in OH and OH+ALT dogs compared with young normal dogs. CML was localized to aortic and aortic vasa vasorum smooth muscle but not to collagen or elastin. CML was essentially undetectable in young normal, OH, or OH+ALT myocardium but was visible in large vessels in the LV. ALT-711 therapy was associated with lower blood pressure and pulse pressure, decreased systemic vascular resistance, increased aortic distensibility and arterial compliance, and, notably, significant aortic dilatation. Neither LV systolic nor diastolic function was different in OH+ALT versus OH dogs.

Conclusions: In elderly hypertensive canines, AGE accumulation and AGE cross-link breaker effects were confined to the vasculature without evidence of myocardial accumulation or effects. The lack of AGE accumulation in collagen-rich areas suggests that the striking vascular effects may be mediated by mechanisms other than collagen cross-linking.

Figure 1. Conscious blood pressure

In elderly dogs subjected to renal wrapping, those treated with ALT-711 (OH+ALT) had lower systolic (SBP) and pulse (PP) pressure than untreated (OH) dogs.

Figure 2. Conscious assessment of arterial properties

Effective arterial elastance (Ea, A) was lower in ALT-711 treated dogs (OH+ALT) with decreases in systemic vascular resistance (SVR, B) and increases in systemic arterial compliance (SAC, C) as compared to untreated (OH) dogs. SAC was inversely related to SVR (p<0.001) and this relationship was similar in treated and untreated animals (D).

Figure 3. Pressure - dimension based assessment of arterial properties in anesthetized animals

Both systolic (A) and diastolic (B) aortic (Ao) area increased with increases in distending pressure (Ao systolic and diastolic pressure (P) respectively) (p<0.001 for both) but were higher in ALT-711 treated dogs (OH+ALT) than untreated dogs at any distending pressure. Aortic wall thickness was lower in OH+ALT dogs (C). The logarithm (Ln) of phasic aortic distensibility (Ao area change (Δ) /pulse pressure (PP); multiplied * 10) decreased with increasing mean Ao P (MAP) (p<0.001) but was higher in OH+ALT than OH dogs at any distending pressure (D).

Figure 4. Pressure - flow based assessment of arterial properties in anesthetized animals

Characteristic aortic impedance calculated with volumetric flow (Zc-flow; A) was lower in ALT-711 treated dogs (OH+ALT) than untreated dogs. Zc calculated with velocity flow tended to be lower in OH+ALT dogs but this was not significant (B). Systemic arterial compliance (SAC) was higher in OH+ALT dogs (C) and the relationship between SAC and systemic vascular resistance (SVR) was shifted upward in OH+ALT dogs (D).

Figure 5. CML content in Aorta

In A, CML staining of aortic tissue (original magnification 10x) from a young normal (YN) dog showing lack of positive staining. In A-D, inserts show higher (20x) magnification of the aortic wall (box) and collagen rich adventitial layer including the vaso vasorum (arrow). In B, CML staining of aortic tissue (10x) from an elderly hypertensive (OH) dog showing dense CML staining throughout the aorta and in the vaso vasorum vascular smooth muscle cells. The CML staining is not associated with the elastin fibrils (pale serpentine appearance here) but rather in smooth muscle cells between the elastin fibrils. In C, picros-sirius staining of sequential section from B showing distribution of collagen (red). The CML staining in B is not in areas of collagen deposition in the media, adventitia or in the vaso vasorum. In D, elastin stain of sequential section from B showing distribution of elastin in relation to CML staining. The CML staining is not associated with elastin. In E, the results of immunohistochemistry CML scoring showing group data with increased score in OH and OH+ALT as compared to YN aorta. In F, group data from Western experiments showing increased CML proportional to GAPDH in OH and OH+ALT as compared to YN aortic tissue. In G, representative Western blot loaded with aortic protein from two YN, five OH and five OH+ALT dog showing staining for CML (≈ 75kDa) and GAPDH (≈ 42kDa).

Figure 6. CML content in Left Ventricle (LV)

CML (A) and picros-sirius (B) staining of sequential LV sections (original10x) in a representative young normal (YN) dog showing lack of CML staining in myocardium or myocardial or peri-vascular collagen. Insert in A-D show higher magnification of myocardium. There is staining in vascular smooth muscle cells. CML (C) and picros-sirius (D) staining of sequential LV sections (10x) in a representative elderly hypertensive (OH) dog showing lack of CML staining in myocardium or myocardial or peri-vascular collagen. There is staining in vascular smooth muscle cells.