Measuring arterial stiffness has recently gained a lot of interest because it is a strong predictor for cardiovascular events and all-cause mortality. However, assessing blood vessel stiffness is not easy and the in vivo measurements currently used provide only limited information. Ex vivo experiments allow for a more thorough investigation of (altered) arterial biomechanical properties. Such experiments can be performed either statically or dynamically, where the latter better corresponds to physiological conditions. In a dynamic setup, arterial segments oscillate between two predefined forces, mimicking the diastolic and systolic pressures from an in vivo setting. Consequently, these oscillations result in a pulsatile load (i.e., the pulse pressure). The importance of pulse pressure on the ex vivo measurement of arterial stiffness is not completely understood. Here, we demonstrate that pulsatile load modulates the overall stiffness of the aortic tissue in an ex vivo setup. More specifically, increasing pulsatile load softens the aortic tissue. Moreover, vascular smooth muscle cell (VSMC) function was affected by pulse pressure. VSMC contraction and basal tonus showed a dependence on the amplitude of the applied pulse pressure. In addition, two distinct regions of the aorta, namely the thoracic descending aorta (TDA) and the abdominal infrarenal aorta (AIA), responded differently to changes in pulse pressure. Our data indicate that pulse pressure alters ex vivo measurements of arterial stiffness and should be considered as an important variable in future experiments. More research should be conducted in order to determine which biomechanical properties are affected due to changes in pulse pressure. The elucidation of the underlying pulse pressure-sensitive properties would improve our understanding of blood vessel biomechanics and could potentially yield new therapeutic insights.

We aimed to explore the role and possible mechanism of leptin in lower-extremity artery calcification in patients with type 2 diabetes mellitus (T2DM).
We recruited 59 male patients with T2DM and 39 non-diabetic male participants. All participants underwent computed tomography scan of lower-extremity arteries. The calcification scores (CSs) were analyzed by standardized software. Plasma leptin level was determined by radioimmunoassay kits. Human vascular smooth muscle cells (VSMCs) calcification model was established by beta-glycerophosphate and calcium chlorideinduction. Calcium deposition and mineralization were measured by the o-cresolphthalein complexone method and Alizarin Red staining. The mRNA expression of bone morphogenic protein 2 (BMP2), runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and osteopontin (OPN) was determined by quantitative RT-PCR. The protein levels of BMP2, Runx2, α-smooth muscle actin (α-SMA) and (p)-Akt was determined by Western-blot analysis, and α-SMA was also measured by immunofluorescence analysis.
Compared with controls, patients with T2DM showed higher median calcification score in lower-extremity artery [286.50 (IQR 83.41, 1082.00) vs 68.66 (3.41, 141.30), p<0.01]. Plasma leptin level was higher in patients with calcification score ≥300 than ≥100 (252.67 ± 98.57 vs 189.38 ± 44.19 pg/ml, p<0.05). Compared with calcification medium, intracellular calcium content was significantly increased in VSMCs treated by leptin (200, 400 and 800 ng/ml) combined with calcification medium [11.99 ± 3.63, 15.18 ± 4.55, and 24.14 ± 5.85 mg/ml, respectively, vs 7.27 ± 1.54 mg/ml, all p<0.01]. Compared with calcification medium, Alizarin Red staining showed calcium disposition was more obvious, and the mRNA level of BMP2, Runx2 and OCN was significantly increased, and immunofluorescence and Western blot analysis showed that the expression of α-SMA was downregulated in VSMCs treated by leptin (400 ng/ml) combined with calcification medium, respectively. Compared with calcification medium, the protein level of BMP2 and Runx2 was upregulated in VSMCs treated by leptin (400 ng/ml) combined with calcification medium. Moreover, blocking PI3K/Akt signaling pathway can decrease the protein expression of BMP2 and Runx2 in VSMCs treated by leptin (400 ng/ml) combined with calcification medium.
Leptin promoted lower-extremity artery calcification of T2DM by upregulating the expression of BMP2 and Runx2, and regulating phenotypic switch of VSMCs via PI3K/Akt signaling pathway.