Introduction:
Severe and symptomatic aortic valve stenosis (AS) has a high mortality of 50% in the course of 2 years. Diabetes mellitus (DM) is a risk factor for the development of aortic valve stenosis. Nevertheless, data on hyperglycemia is controversial. Therefore, we investigated the development of aortic valve stenosis in early stages of diabetic mouse models and the effect of high glucose levels on human valvular interstitial cells (VIC). We hypothesized that early-stage diabetes promotes the development of aortic valve stenosis in vivo. 

Method:
For the in vitro experiments, VIC were exposed to different glucose concentrations for 2-21 days. Cellular gene expression and extracellular matrix calcification were analyzed. 

AS in mice was induced via a wire injury model as previously described. Animals were assigned to four groups of intervention: 1. Type 1 diabetes mellitus (DM1) model: a. High dose streptozotocin (STZ) and wire injury (n=17), b. High dose STZ and sham procedure (n=9), 2. Type 2 diabetes mellitus (DM2) model: a. High-fat diet (DIO 60 %) + low dose STZ and wire injury (n=10), b. High-fat diet (DIO 60 %) + low dose STZ and sham procedure (n=10), 3. diet control: a. DIO 60 % and wire injury (n=10), b. DIO 60 % and sham procedure (n=10) and 4. wire injury control (n=10). Body parameters were measured weekly. Glucose tolerance tests were obtained via an i.p. glucose injection. Echocardiography was obtained every two weeks over 6 weeks after surgery. Hearts were analysed histologically by staining (i.a. HE, von Kossa).                   

Results:
Incubation of VICs in procalcifying medium with elevated glucose concentrations promoted both enhanced calcification as shown by Alizarin red staining and overexpression of RUNX2 as well increased inflammation as demonstrated by IL-1b expression.

Diabetic mice showed pathological glucose tolerance tests (glucose in mg/dl after 0-15-30-60-120 minutes: AS-DM1 382-698-573-526-421, sham-DM1 375-555-583-480-400, AS-DM2 221-518-531-421-258, sham-DM2 179-406-422-342-227, AS-DIO60 179-380-346-251-172, sham-DIO60 217-419-384-256-164, AS-Co 164-299-257-213-173). While mice receiving a high-fat diet rapidly increased body weight,  mice treated with high doses of STZ showed significantly lower weight gain (body weight in g (week 6): AS-DM1 26.3, sham-DM1 25.7, AS-DM2 29.7, sham-DM2 28.8, AS-DIO60 29.3, sham-DIO60 28.9, AS-Co 27.3).

Six weeks after wire injury, mice developed significant AS, as illustrated by increased peak blood flow velocity over the aortic valve. There were however no significant differences between the wire injury intervention groups (peak velocity (week 6) in mm/s: AS-DM1 1884, AS-DM2 2106, AS-DIO60 1978 vs AS-Co 1980, p=ns). Sham groups showed no change in peak blood flow velocity (peak velocity (week 6) in mm/s: sham-DM1 1199, sham-DM2 1155, sham-DIO60 1109, p=ns).

Histologically, the aortic valve cusp tissue area was similarly enlarged in all treatment groups (AS-DM1: 176,099 µm²; AS-DM2 133,779 µm²; AS-DIO60 149,850 µm²; vs AS-Co: 138,802 µm², p=ns). Interestingly, increased calcification could be detected in diabetic and high-fat fed mice (percent of area: AS-DM1 4.8; AS-DM2 5.4; AS-DIO60 4.6; AS-Co 2.2; p<0.05).

Conclusion:
Although in vitro data suggest elevated glucose levels promote mechanisms associated with AS formation, AS development is similar in diabetic and control mice. Further studies have to be executed in order to evaluate a more long-term effect of DM in this model of AS.