Effect of Lysyl Oxidase Inhibition on Angiotensin II-Induced Arterial Hypertension, Remodeling, and Stiffness

<div><p>It is well accepted that angiotensin II (Ang II) induces altered vascular stiffness through responses including both structural and material remodeling. Concurrent with remodeling is the induction of the enzyme lysyl oxidase (LOX) through which ECM proteins are cross-linked. The study objective was to determine the effect of LOX mediated cross-linking on vascular mechanical properties. Three-month old mice were chronically treated with Ang II with or without the LOX blocker, β -aminopropionitrile (BAPN), for 14 days. Pulse wave velocity (PWV) from Doppler measurements of the aortic flow wave was used to quantify in vivo vascular stiffness in terms of an effective Young’s modulus. The increase in effective Young’s modulus with Ang II administration was abolished with the addition of BAPN, suggesting that the material properties are a major controlling element in vascular stiffness. BAPN inhibited the Ang II induced collagen cross-link formation by 2-fold and PWV by 44% (P<0.05). Consistent with this observation, morphometric analysis showed that BAPN did not affect the Ang II mediated increase in medial thickness but significantly reduced the adventitial thickness. Since the hypertensive state contributes to the measured in vivo PWV stiffness, we removed the Ang II infusion pumps on Day 14 and achieved normal arterial blood pressures. With pump removal we observed a decrease of the PWV in the Ang II group to 25% above that of the control values (P=0.002), with a complete return to control values in the Ang II plus BAPN group. In conclusion, we have shown that the increase in vascular stiffness with 14 day Ang II administration results from a combination of hypertension-induced wall strain, adventitial wall thickening and Ang II mediated LOX ECM cross-linking, which is a major material source of vascular stiffening, and that the increased PWV was significantly inhibited with co-administration of BAPN.</p></div

Effect of Ang II and BAPN on LOX, collagen, and cross-linking in aortas.

<p>The effect of LOX is not confined to the crosslinking of collagen and elastin and therefore we examined the effect of LOX on gene expression of other key ECM proteins and enzymes. <b>A</b>, Lysyl-oxidase enzymatic activity measured from the lower thoracic aorta represented by the production of H₂O₂ and detected by Amplex red oxidation. <b>B</b>, The total collagen content using hydroxyproline concentrations assuming collagen contains 13.5% hydroxyproline. <b>C</b>, Cross-linked collagen, using cyanogen bromide as digestion. <b>D</b>, Percent of collagen cross-linking. n = 6. *P<0.05 compared to control, ^ƗP<0.05 compared with BAPN group, ^#P<0.05 compared with Ang II group at their respective day.</p

Cessation of Ang II infusion.

<p>Hypertension contributes to vascular stiffness together with structural and material remodeling. Therefore the Alzet pumps were removed to determine the contribution of each to the total vascular stiffness. <b>A</b>, The Alzet pumps were removed on Day 14 and the blood pressure returned to control values by day + 4. <b>B</b>, The pulse wave velocity measured with and without Ang II infusion on days 14 and + 4 days after pump removal. n = 6. *P<0.05 compared to control, ^ƗP<0.05 compared with Ang II group, ^#P<0.05 compared with Ang II + BAPN + 4 group.</p

Vascular morphology.

<p>Aortic histomorphometry was performed with the elastin specific stain Verhoeff’s Van Gieson (VVG) and the collagen specific stain picrosirius red (PSR). <b>A</b>, Representative histological sections are presented according to treatment. <b>B</b>, The medial thickness and adventitial thickness were analyzed to describe the aortic morphological changes in response to the treatments. <b>C</b>, The aortic structural dimensions as measured with VVG and PSR stains. <b>D</b>, The systolic luminal diameter were measured with the M-mode ECHO, collagen content was quantified with PSR histological staining, and elastin content was measured with VVG stain. n = 6. *P<0.05 compared to control, ^ƗP<0.05 compared with BAPN group.</p

Aortic flow and stiffness characteristics.

<p>The flow characteristics of the lower thoracic aorta, superior to the renal bifurcation, were analyzed with ECHO/Doppler. <b>A</b>, Pulse wave velocity was used to determine vascular stiffness. <b>B</b>, Effective wall stiffness (Eh), was deduced from measuring PWV. <b>C</b>, Wall thickness (h), was measured from perfusion fixed aortas at day 14, at the same location of the PWV readings. <b>D</b>, The effective Young’s modulus (E), readings were made weekly. <b>E</b>, The diastolic flow percentages was calculated comparing the systolic and diastolic VTI’s. <b>F</b>, The reflected wave transit time was measured at the innominate artery with flow Doppler. n = 6. *P<0.05 compared to control, ^ƗP<0.05 compared with BAPN group, ^#P<0.05 compared with Ang II group at their respective day.</p