Introduction
Osteoporosis is a common skeletal condition that affects millions of patients worldwide. In recent decades, etiological studies of osteoporosis were mostly focused on the increased osteoclast functions or the decreased osteoblast functions. More recently, vessel abnormalities have become a new research area for osteoporosis. Kusumbe et al. found that type H vessels in a specific murine skeletal system could mediate the growth of the bone vasculature and generate distinct metabolic and molecular microenvironments, as well as maintain perivascular osteoprogenitors and couple angiogenesis to osteogenesis [
1]. Molecular frameworks were also identified to couple angiogenesis, angiocrine signals, and osteogenesis [
2,
3]. In humans, studies show that type H vessels are upregulated in aged and osteopenia patients [
4]. These findings suggest a link between the vasculature and osteoporosis; however, these human bone samples were collected from patients with hip fractures, which required surgical intervention [
4]. More clinical evidence is required to clarify the relationship between vascular abnormalities and osteoporosis.
Type 2 diabetes mellitus (T2DM) is often complicated by vascular diseases. Patients with T2DM had a high risk for peripheral vascular disease, including carotid artery stenosis and lower extremity peripheral artery disease [
5]. In our previous studies [
6,
7], we demonstrated the association between diabetic vascular diseases (microangiopathy and macroangiopathy) and the presence of osteoporosis. Patients with the highest 24-h urine protein levels had the lowest bone mineral density (BMD) in both sexes [
6]. Multivariate analysis revealed that diabetic retinopathy was negatively related to BMD in women, but not in men [
6]. In addition, we identified a negative correlation between carotid plaque scores and BMD in women with T2DM. A high carotid plaque score with calcification might be a risk factor for osteoporosis [
7]. However, research on the relationship between lower extremity arteries and BMD remains limited.
In our previous study, we investigated the relationship between macroangiopathy and BMD in different groups of T2DM patients, including men vs. women, obese vs. non-obese individuals, hypertension vs. non-hypertension, hyperlipidemia vs. non-hyperlipidemia, and calcification vs. non-calcification groups. We found that only sex and calcification were relevant [
7]. Therefore, we assumed that the relationship between lower extremity macroangiopathy and BMD could depend on sex and calcification. In the present study, we evaluated the sex and calcification differences in the relationship between lower extremity arteries and the presence of osteoporosis in T2DM patients.
Materials and methods
Study design and participants
We conducted a retrospective cross-sectional study. Patients with T2DM who visited the inpatient clinic of the Department of Endocrinology and Metabolism, Shanghai Tenth People’s Hospital, Tongji University School of Medicine (China), from July 2018 to February 2020, were reviewed in this study. Study participants were selected based on the inclusion and exclusion criteria. This study was approved by the Ethics Committee of the hospital. The requirement for informed consent was waived due to the retrospective design of the study.
Inclusion and exclusion criteria
Inclusion criteria were (1) newly diagnosed T2DM patients or T2DM patients with diabetes-associated complications (e.g., uncontrolled blood glucose, diabetic neuropathy, diabetic nephropathy, and diabetic retinopathy). Diabetes mellitus was diagnosed by either fasting plasma glucose (FPG) (≥ 7.0 mmol/l), 2-h plasma glucose (2-h PG) concentration (≥ 11.1 mmol/l) after a 75-g oral glucose tolerance test (OGTT) [
8], or a history of diabetes. T2DM was diagnosed based on the serum insulin or C-peptide level and diabetic-associated antibodies (glutamic acid decarboxylase antibody, insulin autoantibody, and islet cell antibody) [
9]; (2) patients with complete data of color Doppler ultrasonography of lower extremity arteries and dual-energy X-ray absorptiometry (DXA) for BMD. Exclusion criteria were (1) male patients < 50 years old or premenopausal female patients (these two groups were excluded due to their small sample sizes); (2) patients with severe end-stage renal disease (a history of renal dysfunction or clearance of creatinine less than 30 ml/min), liver disease (a history of liver dysfunction or alanine aminotransferase more than 100 U/L), or cardiac issues (a history of heart dysfunction or ejection fraction less than 50%); (3) patients with a history of secondary osteoporosis or those diagnosed after routine examination; (4) patients on medications that could influence bone metabolism, including bisphosphonates, calcitonin, selective estrogen receptor modulators (SERMs), estrogens, or glucocorticoid. For patients with repeat hospital visits, we only analyzed their data collected during the first visit.
Sample size was calculated according to the formula for a cross-sectional study (N = Z21-α*p*(1-p)/d2). The study required more than 189 men (Z1-α = 1.96, p = 0.144, d = 0.05) and 124 women (Z1-α = 1.96, p = 0.207, d = 0.05).
Demographic variables
Medical records on age, sex, and T2DM duration were collected. Blood pressure, height, and weight were documented. Pulse pressure (an index of arteriosclerotic stiffness and impaired arterial conduit according to Windkessel physiology) was calculated as the systolic blood pressure minus the diastolic blood pressure. Body mass index (BMI, kg/m2) was also calculated. Information on current medications and menopausal status for women was collected from the electronic and paper medical records.
Laboratory measurements
Laboratory test results were collected from electronic medical records. The methods of testing were as follows. Fasting blood was collected after overnight fasting. Alanine aminotransferase (ALT), serum creatinine (SCr), and serum calcium and phosphorus were analyzed by a routine auto-analyzer (Modular DP analyzer, Roche Diagnostics, Mannheim, Germany). Total cholesterol (TC) and triglycerides (TG) were determined by the enzyme-linked immunosorbent assay (ELISA) method. Clearance of creatinine (CLCR) was calculated by the Cockcroft-Gault formula (men: CLCR = ((140-age [years]) * body weight [kg])/(0.818*SCr [μmol/l]); women: CLCR = ((140-age [years]) * body weight [kg]) * 0.85)/(0.818*SCr [μmol/l]). Hemoglobin A1 c (HbA1c) was determined by high-performance liquid chromatography (HPLC) with a coefficient of variation (CV) of 1.11%. Serum fasting C-peptide (CP), osteocalcin, C-terminal telopeptide fragments of type I collagen (β-CTX), N-terminal propeptide of type I procollagen (PINP), bone alkaline phosphatase (BAP), parathyroid hormone (PTH), and 25 (OH) vitamin D were measured using radioimmunoassay (RIA).
Ultrasonography of the lower extremity arteries
Ultrasonography test results were collected from electronic medical records. The methods of testing were as follows. Lower extremity arteries, including the common femoral artery, superficial femoral artery, popliteal artery, and dorsal artery of the foot, were assessed by high-resolution B-mode and color Doppler/pulsed-wave Doppler ultrasonography (GE LO5, LOGIQ 5, USA) with a transducer frequency of 7.5 MHz. Plaques were identified by the hyperechogenic images inside the artery. Calcification was identified by the bright white echoes protruding into the lumen. The study participants were assigned into four groups based on their calcification status and sex.
Bone mineral density
Values for the areal BMD (g/cm
2) at the lumbar spine (L1–L4), hip, and femoral neck were obtained by DXA on a Hologic QDR 4500 W densitometer with software version 9.03 (Hologic, USA). All examinations were performed on one device and by one technologist on the same settings following standardized procedures. The BMD devices provided absolute values for the lumbar spine, hip, and femoral neck, along with T-scores or Z-scores. The osteoporosis diagnostic criteria [
10] from the World Health Organization in postmenopausal women and men aged ≥ 50 years were based on the T-score. Normal BMD was defined as a T-score ≥ − 1.0. Osteopenia was defined as a T-score from > − 2.5 to < − 1.0. Osteoporosis was defined as a value for T-score ≤ − 2.5 SD in any of the three sites.
Statistical analysis
Normally distributed continuous data are presented as means ± SD. Non-normally distributed continuous data are presented as medians and interquartile range (IQR). Categorical data are presented as percentages. To compare the patients with or without vascular calcification in both sexes, independent t test or Mann-Whitney U test was performed when appropriate. A chi-square test was conducted to compare the difference of osteoporosis between patients with or without calcification. The relationships of lower extremity arteries with prevalence of osteoporosis were further analyzed by ordinal logistic regression analysis. The dependent variables were normal BMD, osteopenia, and osteoporosis. The independent variables that were included in the regression analysis were based on the clinical significance and statistical significance (bivariate analysis with a P < 0.2). Potential confounding factors included age, the duration of T2DM, BMI, pulse pressure, CLCR, glycosylated hemoglobin, and fasting C-peptide. ORs and corresponding 95% confidence intervals are reported. Statistical analysis was conducted in SPSS 26.0 (SPSS Inc., Chicago, IL, USA). A two-sided P < 0.05 was considered statistically significant.
Discussion
In this study, we found that lower extremity arteries with calcification doubled the risk for osteoporosis in T2DM women after adjusting for several other risk factors (i.e., age, the duration of T2DM, body mass index, pulse pressure, clearance of creatinine, glycosylated hemoglobin ,and fasting C-peptide), not in men. These findings are consistent with the hypothesis that lower extremity arteries are associated with osteoporosis depending on calcification and sex. Studies on the relationship between lower extremity arteries and bone are limited. However, there is some evidence indicating a relationship between atherosclerosis calcification and BMD. For example, Alan et al. found a significant association between cardiovascular disease and low bone mass [
11]. BMD is a promising marker for the severity of coronary lesions [
12] and coronary artery calcium [
13]. Previous studies including ours [
7] have suggested that calcification, not plagues, is associated with low BMD and higher risk for osteoporosis. The mechanism was considered to be the effects of chronic inflammation and/or the oxidative stress of calcium metabolism [
11‐
13]. Risk factors, such as aging and decreased levels of sex hormones (especially estrogen), could lead to increased levels of inflammatory and oxidative stress cytokines [
14,
15]. These cytokines, such as tumor necrosis factor-α and interleukin-1β, could increase the level of receptor activator of nuclear factor kappa β ligand (RANKL), which further activates osteoclasts and results in a shift of calcium from bones to the vessel walls [
16]. This shift of calcium might lead to calcification of atherosclerosis and low BMD. Calcification forms specifically in the muscle cells adjacent to atheromas as well as on the surface of atheroma plaques and tissues. However, in this study, differences in the bone metabolic markers were not found to be significant. It is possible that the change in bone turnover markers was blunted in diabetes mellitus [
17]. It should be mentioned that medial artery calcification (MAC) is another type of arterial calcification different from atherosclerosis with calcification. MAC, also known as Mönckeberg’s arteriosclerosis, is a condition that leads to the stiffening of the elastic layer of the arterial wall. It is also a common feature of conduit artery disease in T2DM [
18]. However, in contrast to intimal arterial calcification, MAC did not obstruct the arterial lumen [
19]. In the current study, we did not report MAC in the recruited T2DM patients by ultrasonography. The reason for not reporting MAC is that MAC is better identified by radiogram, whereas atherosclerosis calcification is commonly detected by ultrasonography [
20]. In this retrospective study, our patients only received an ultrasonography examination. Moreover, MAC was more frequently observed in patients with diabetic nephropathy, particularly those on hemodialysis [
20‐
22]. In our study, patients with impaired renal function were excluded from the analysis. In the future, we plan to perform prospective studies to independently assess atherosclerosis vs. medial calcification and their relationships with osteoporosis risk.
We found that the correlation between lower extremity arterial calcification and osteoporosis only existed in postmenopausal women, not in men, for unclear reasons. Similar sex differences in association between vascular diseases and BMD have previously been reported in our studies [
6,
7]. Other research has also shown that postmenopausal women with vascular diseases were vulnerable to osteoporosis [
23‐
25]. This might be because postmenopausal women, as compared to men, have a high risk to develop osteoporosis. It is worth mentioning that, in a previous longitudinal study (MrOS), Cummings and colleagues [
26] demonstrated high rates of hip bone loss and increased fracture risk in older men with peripheral arterial disease. The participants in the MrOS study were community-dwelling who had a lower prevalence of diabetes mellitus. They were grouped by peripheral artery disease as measured by ankle-brachial index (ABI). We speculate that different study populations, different testing methods, and different groupings led to the results different from ours. Sex differences might be attributable to sex hormones, though this requires further study.
To the best of our knowledge, these data are the first to demonstrate a relationship between lower extremity arterial calcification and the presence of osteoporosis. Interestingly, we found that lower extremity arterial calcification was related with lower BMD of the hip and femur neck. The BMD of the lumbar spine was not different between groups with or without calcification. This is different from the relationship between carotid arterial calcification and BMD, which is related with lower BMD of lumbar spine, hip, and femur neck [
7]. The lower extremity arteries include the common femoral artery, superficial femoral artery, popliteal artery, and dorsal artery of the foot. These four arteries provide blood to the hip and femoral neck. The lumbar spine is supplied with blood by the lumbar arteries, which were not examined in this study. The calcification of lumbar arteries was thus not clear. It might be that lower extremity arterial calcification is more closely associated with the BMD of the hip and femur neck. Moreover, the BMD of the lumbar spine might also be related to age, weight, BMI, or estradiol [
27,
28]. Nevertheless, we found that the prevalence of osteoporosis was higher in patients with calcification in women.
Notably, the relationship between vascular disease and abnormal BMD is not the same in microangiopathy and macroangiopathy. In our previous study, the severity of microangiopathy was related to low BMD in women T2DM patients [
6]. However, in the current study, a relationship between macroangiopathy without calcification and BMD was not identified. This might indicate that macrovascular disease and microvascular disease affect osteoporosis by different mechanisms. We inferred that macrovasculature results in the infusion of bone; however, microvasculature not only provides nutrients to the bone but also adjusts bone health [
8,
29]. Thus, more studies should be performed to investigate the role of vasculopathy in the process of osteoporosis.
In addition to the inherent biases in the retrospective investigation, there are other limitations in the present study. First, there was a lack of data on bone fractures. We will perform additional studies to collect fracture information in future research. Second, cause-and-effect relationships between arterial calcification and osteoporosis could not be determined from the present study due to the cross-sectional study design. Third, since the main complaints of these patients visiting the hospital were for diabetes mellitus instead of osteoporosis, factors that could potentially affect osteoporosis, such as nutritional status (e.g., calcium and protein intake) and weight-bearing physical activity, were not recorded for the analysis. Fourth, granularity of the data on lower extremity arterial calcification, such as details on calcification of each artery, number of vascular beds with calcification, and the extent of calcification, could not be traced in this retrospective observational study. Finally, we performed this study in patients with T2DM, which limited the generalizability of our study results to patients without this disease.
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