• Users Online: 76
  • Home
  • Print this page
  • Email this page
Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login 


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2013  |  Volume : 40  |  Issue : 4  |  Page : 211-223

Role of vascular endothelial growth factor expression in pathogenesis of postmenopausal osteoporosis


1 Department of Physical Medicine, Rheumatology and Rehabilitation, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Orthopaedic Surgery, Faculty of Medicine, Ain Shams University, Cairo, Egypt
3 Department of Histology, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Date of Submission20-Apr-2013
Date of Acceptance12-Jul-2013
Date of Web Publication30-Dec-2013

Correspondence Address:
Eman A Kaddah
Department of Physical Medicine, Rheumatology, and Rehabilitation, Faulty of Medicine, Ain Shams University, Cairo
Egypt
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-161X.123809

Rights and Permissions
  Abstract 

Background
Vascular endothelial growth factor (VEGF), an angiogenic growth factor, has been proved to play a significant role in bone remodeling. It may be involved in the molecular pathogenesis of postmenopausal osteoporosis.
Aim
The aim of this study was to investigate the expression of VEGF in bone biopsies of postmenopausal osteoporotic patients, assess the relation between the expression of VEGF and bone mineral density (BMD), and to evaluate the association between VEGF, serum estradiol, and bone estrogen receptor-α.
Patients and methods
This study was carried out on 30 female patients who were further subdivided into three groups: premenopausal, perimenopausal, and postmenopausal. All of them were subjected to full assessment of history, thorough clinical examination, and routine laboratory investigations. Serum estradiol levels were measured using ELISA. BMD was detected using DEXA. Bone biopsies were taken and three sections were obtained from each specimen. One was stained with hematoxylin and eosin stain for bone histomorphometrical assessment. The other two sections were stained immunohistochemically for the detection of VEGF and estrogen receptor-α (ER-α) expression.
Results
A highly statistically significant difference was found in VEGF expression between the premenopausal, perimenopausal, and postmenopausal women and also between osteoporotic and nonosteoporotic women. A highly statistically positive correlation was found between VEGF and each of the following: BMD, bone anabolic histomorphometrical parameters E2, and ER-α. However, a highly statistically negative correlation was observed between VEGF and bone histomorphometrical resorption parameters.
Conclusion
VEGF expression is decreased in bone of postmenopausal osteoporotic patients and is correlated to BMD. Its release is dependent on E2 and mediated through ER-α. These suggest that bone alterations induced by reduced estrogen in postmenopausal osteoporosis may be partly through decreased VEGF release. This makes it one of the possible targets in the treatment of postmenopausal osteoporosis.

Keywords: bone mineral density, postmenopausal osteoporosis, vascular endothelial growth factor


How to cite this article:
Nasser ME, Khaled HF, Kaddah EA, Elbadrawy AM, Mahdi SM, Sharobeem MA. Role of vascular endothelial growth factor expression in pathogenesis of postmenopausal osteoporosis. Egypt Rheumatol Rehabil 2013;40:211-23

How to cite this URL:
Nasser ME, Khaled HF, Kaddah EA, Elbadrawy AM, Mahdi SM, Sharobeem MA. Role of vascular endothelial growth factor expression in pathogenesis of postmenopausal osteoporosis. Egypt Rheumatol Rehabil [serial online] 2013 [cited 2017 Dec 13];40:211-23. Available from: http://www.err.eg.net/text.asp?2013/40/4/211/123809


  Introduction Top


Osteoporosis is a progressive systemic skeletal disorder characterized by low bone mineral density (BMD) and microarchitectural deterioration of bone tissue that reduces bone strength and increases the risk of fractures [1] .

Postmenopausal osteoporosis is mainly caused by increased bone remodeling resulting from estrogen deficiency with induced imbalance between bone formation and resorption such that resorption is favored over formation [2] .

Bone biopsies from postmenopausal osteoporotic patients were characterized by a reduction in sinusoidal and arterial capillaries in the bone marrow (BM) and reduced bone perfusion, suggesting the role of a vascular component in the pathogenesis of this disease and confirming what was reported previously, that is, coupling between angiogenesis and osteogenesis was essential for normal bone formation [3] .

Vascular endothelial growth factors (VEGFs) and their corresponding receptors are key regulators in a cascade of molecular and cellular events that ultimately lead to angiogenesis [4] . Although the main effects of VEGFs are on endothelial cells, they also bind to VEGF receptors expressed on monocytes, neurons, chondrocytes, and osteoblasts [5] .

VEGF is known to recruit endothelial cells and the endothelial cells themselves organize bone remodeling. Once recruited, endothelial cells release a potent mitogen for osteoblasts. However, endothelial cells could also inhibit and regulate osteoclast activity [6] . A previous study [7] reported that VEGF exerted a direct effect on osteoprogenitor cells by promoting their differentiation into osteoblasts. It could also promote mineralization of the bone and increase the bone density. Later, it was observed that osteoblasts themselves released VEGF in an autocrine manner to regulate their own activity. Furthermore, this expression of VEGF in osteoblasts was regulated by estrogens such as 17 b-estradiol [8] .

Activated estrogen receptors (ERs) have been reported to induce hypoxia inducible factor-1 α (HIF-1α) activation, which stimulates VEGF-mediated angiogenesis in bone [9] . Vitamin D3 was proved to increase VEGF expression, suggesting that the anabolic effects of vitamin D on bone tissue may be partly mediated by VEGF [10] .

A possible association between VEGF activity and the pathophysiology of osteoporosis was suggested by Pufe et al. [11] , who reported that the decrease in VEGF levels played a role in glucocorticoid-induced osteoporosis in experimental animals. A few years later, it was documented that the reduction in the BMD in the lumbar spines of ovariectomized experimental animals correlated with the decrease in VEGF levels [6] .

It has been reported that after menopause, there is a decrease in the levels of VEGF [12] . It is also worth mentioning that hormone replacement therapy increased VEGF in postmenopausal women [6] . To our knowledge, the expression of VEGF in bone biopsies of postmenopausal osteoporotic patients and its correlation with their BMD have not been studied before.


  Aim of the work Top


The aim of this work was to investigate the expression of VEGF in bone biopsies of postmenopausal osteoporotic patients, assess the relation between the expression of VEGF and BMD in these patients, and to evaluate the association between VEGF, serum estradiol, and bone ER-α in order to detect a role of VEGF in the pathogenic pathway of postmenopausal osteoporosis, which, if proved, could potentially be a therapeutic target.


  Patients and methods Top


This study included 30 female patients who attended the Orthopedic and/or the Physical Medicine, Rheumatology, and Rehabilitation Departments of Ain Shams University Hospitals. All were candidates for operations during which an iliac crest bone biopsy was taken from each patient after obtaining an informed written consent and after the study had already been approved by the Ethics Committee of the Faculty of Medicine, Ain Shams University. Patients were divided into three groups according to menopausal status: Premenopausal, perimenopausal, and postmenopausal after the study by Schmidt and Rubinow [13] , who defined perimenopausal women as those who had menstrual irregularities and menopause as the permanent cessation of menstruation for 12 months.

Exclusion criteria

Patients with secondary osteoporosis because of any cause such as rheumatological, connective tissue, endocrinal, chronic renal failure, immobilization, and drug-induced osteoporosis were excluded as well as patients receiving medical treatment for osteoporosis or hormone replacement therapy.

All patients were subjected to the following:

  1. Full assessment of history, with a special focus on menstrual history, drug intake, history of generalized bone aches, or nontraumatic fractures.
  2. Thorough clinical examination that included height and weight measurements to calculate BMI. General, spine, neurological, and joint assessment was performed.
  3. Laboratory investigations included the following:
    1. Complete blood count using a Coulter counter.
    2. Erythrocyte sedimentation rate (ESR) using the Westergren method.
    3. Fasting blood sugar.
    4. Liver function tests: Aspartate aminotransferase and alanine aminotransferase.
    5. Renal function tests: Serum creatinine and urea.
    6. Serum calcium, phosphorous, and alkaline phosphatase.
    7. Serum estradiol (E2) using the enzyme linked immunosorbant assay (ELISA) technique.
  4. Radiological investigations included the following:
    1. Plain radiograph: Anteroposterior and lateral views of the dorsolumbar spine to detect any vertebral deformities or fractures.
    2. Dual-energy X-ray absorptiometry (DEXA): BMD was measured at three sites, the femoral neck, lumbar spine, and distal radius, using the LUNAR (DPX-MD+) device (GE Medical System).
  5. Bone sample: The bone specimens from the iliac crest graft were fixed using 10% neutral-buffered formalin (NBF). Then, they were decalcified using a Ca-chelating agent EDTA. The time required for decalcifying bone was 6-8 weeks. The fluid was changed every 4 days. The specimens were then dehydrated sequentially in ascending concentrations of ethanol (70, 90, and 100%). Next, they were cleared in xylene and embedded in paraffin. Three formalin-fixed paraffin-embedded sections were obtained from each specimen: One section was stained with hematoxylin and eosin (H&E) for histomorphometrical assessment of bone and the other two sections were stained immunohistochemically for the detection of VEGF and ER-α expression.
    1. Histomorphometrical assessment of bone: It is a quantitative method for the evaluation of trabecular bone. An image analysis system Leica Q 500 MC analyzer program was used. The following parameters were measured:



    2. Immunohistochemistry (IHC) for VEGF: Sections were deparaffinized in xylene for 1 h, rehydrated in descending concentrations of alcohol, and blocked with hydrogen peroxide. Then, they were incubated with a primary anti-VEGF, which is a mouse-anti-human monoclonal antibody IgG (Thermo Fisher Scientific, Fremont, California, USA). This was detected by incubation with a biotinylaned secondary anti-mouse antibody. The complexes were visualized after the addition of diaminobenzidine (DAB).
    3. IHC for ER-α: Sections were deparafinized, rehydrated, and blocked as reported previously. Then, they were incubated with a primary anti-ER, which was a rabbit anti-human antibody (Thermo Fisher Scientific). This was detected by incubation with a biotinylaned secondary anti-rabbit antibody. The complexes were visualized after the addition of DAB.


Statistical analysis

Statistical analysis was carried out using the statistical package for social sciences software (SPSS 15.0.1; SPSS Inc.). Quantitative variables (clinical, laboratory, radiological, histomorphometrical parameters of bone, and immunohistochemistry) were presented as mean±SD. The analysis of variance (ANOVA) test was used to compare the means of more than two studied groups. Relationships between parameters were analyzed using Pearson's correlation coefficient. A P value less than 0.05 was considered significant.


  Results Top


This study was carried out on 30 female patients who were divided according to their menopausal status into three groups, each including 10 patients: Premenopausal women were included in group I, perimenopausal women were included in group II, and postmenopausal women were included in group III. Comparison between descriptive and laboratory data of the three groups using the ANOVA test is shown in [Table 1].
Table 1: Comparison of descriptive and laboratory data of patients of the three groups

Click here to view


Comparison of the three groups in serum E2 using the post-hoc test showed a highly statistically significant difference between group I and group II, group I and group III, and group II and group III as shown in [Figure 1].
Figure 1: A bar chart showing a comparison of the three groups in serum estradiol (E2) (pg/ml)

Click here to view


Radiological data

On examining the lateral view radiograph of the dorsolumbar spines, no abnormalities were detected in both groups I and II. However, in group III, four patients (40%) showed both kyphosis and compression fractures in the dorsal spine.

DEXA studies

All women in group one showed BMD within normal ranges, whereas in group II, four women had normal BMD, five women were osteopenic, and only woman was osteoporotic. In group III, all 10 postmenopausal patients were osteoporotic. Comparison of the three groups indicated a statistically significant difference in the T-score at the femoral neck, lumbar spine, and distal forearm as can be seen in [Table 2] and [Figure 2].
Figure 2: A bar chart showing a comparison of the three groups in the T-score at the femoral neck, lumbar spine, and forearm

Click here to view
Table 2: Comparison of the three groups in the T-score at the femoral neck, lumbar spine, and forearm

Click here to view


Histological results

Light microscopic examination of trabecular bone of iliac crest biopsies stained with H&E showed that in group I, continuous thick bone trabeculae separated by BM spaces could be observed as shown in [Figure 3] and [Figure 4].
Figure 3: A photomicrograph of a section from iliac crest bone of a premenopausal woman showing branching and anatomizing thick trabeculae with bone marrow spaces (BM) in between (H&E stain, ×400)

Click here to view
Figure 4: A photomicrograph of a section from iliac crest bone of a premenopausal woman showing the cancellous bone trabeculae and bone marrow spaces (BM). Bone trabeculae are lined with cuboidal osteoblasts (Ob) with rounded nuclei. Osteocytes (Os) inside their lacunae in between the lamellae can also be seen (H&E stain, ×400)

Click here to view


In group II, iliac crest bone biopsies showed slightly discontinuous bone trabeculae separated by widened BM spaces. Larger erosion cavities were detected in comparison with premenopausal biopsies as shown in [Figure 5].
Figure 5: A photomicrograph of a section from iliac crest bone of a perimenopausal woman showing slightly discontinuous bone trabeculae that appeared relatively thin separated by widened bone marrow spaces (BM) (H&E stain, ×400)

Click here to view


In group III, bone trabeculae appeared significantly discontinuous and thin. They were separated by markedly widened BM spaces containing abundant fat cells. The largest erosion cavities were as frequently detected as shown in [Figure 6] and [Figure 7].
Figure 6: A photomicrograph of a section from iliac crest bone of a postmenopausal woman showing significantly discontinuous thin bone trabeculae separated by widened bone marrow spaces (BM) containing abundant fat cells (H&E stain, ×400)

Click here to view
Figure 7: A photomicrograph of a section from iliac crest bone of a postmenopausal woman showing an irregular eroded surface (ES) of bone trabeculae (H&E stain, ×400)

Click here to view


Histomorphometrical assessment of bone biopsies

Comparison of the three groups in the histomorphometrical parameters of bone that included BV, TbTh, ObS, TS, and ES using the ANOVA test showed a highly statistically significant difference (P < 0.001) between them all as shown in [Table 3] and [Figure 8] and [Figure 9].
Figure 8: A bar chart showing a comparison of the three groups in BV, TbTh, and ObS. BV, bone volume; ObS, osteoblast surface; TbTh, trabecular bone thickness.

Click here to view
Figure 9: A bar chart showing comparison of the three groups in TS and ES. ES, eroded surface; TS, trabecular separation

Click here to view
Table 3: Comparison of the three groups in the histomorphometrical parameters of bone

Click here to view


Immunohistochemical results for VEGF expression

Cytoplasmic immune-labeling for VEGF appeared as brownish granules or aggregates within the cytoplasm.

In premenopausal patients of group I, bone biopsies showed enhanced VEGF expression. Many osteogenic cells, osteoblasts lining trabeculae, and BM stromal cells showed an intensely positive cytoplasmic reaction, with the percentage of VEGF expression ranging from 3.87 to 9.39%, mean of 7.23 ± 1.94%, as shown in [Figure 10].
Figure 10: A photomicrograph of a section from iliac crest bone of a premenopausal patient showing cytoplasmic reaction of the vascular endothelial growth factor expression in the osteoblasts (IHC stain, ×400)

Click here to view


In perimenopausal patients of group II, there was decreased VEGF expression (ranging from 1.40 to 2.78%, mean 1.98 ± 0.48%) in fewer osteogenic, osteoblasts, and BM stromal cells compared with group I as shown in [Figure 11]
Figure 11: A photomicrograph of a section from iliac crest bone of a perimenopausal woman shows decreased vascular endothelial growth factor expression in fewer osteoblasts (IHC stain, ×400)

Click here to view


In postmenopausal patients of group III, VEGF expression was almost absent and was only found in very few and scattered cells (expression percentage ranged from 0.13 to 1.22%, mean 0.64 ± 0.37%) as shown in [Figure 12]
Figure 12: A photomicrograph of a section from iliac crest bone of a postmenopausal woman showing very minimal vascular endothelial growth factor expression in osteoblasts lining bone trabeculae and stromal cells (IHC stain, ×400)

Click here to view


Immunohistochemical results for ER-α expression

ER-α expression appeared as both nuclear and cytoplasmic immunolabeling, which consisted of brownish granules or aggregates of variable intensity in osteoblasts, osteogenic cells, and BM stromal cells.

In group I, enhanced ER-α expression was observed (ranging from 3.93 to 7.89%, mean 6.00 ± 1.64%) as shown in [Figure 13] and [Figure 14].
Figure 13: A photomicrograph of a section from iliac crest bone of a premenopausal woman showing an enhanced estrogen receptor-α (ER-α) expression in osteoblasts lining bone trabeculae that contained brownish ER-α-positive granules (IHC stain, ×400)

Click here to view
Figure 14: A photomicrograph showing bone marrow (BM) spaces of a premenopausal woman with estrogen receptor-α positively stained BM cells (IHC stain, ×400)

Click here to view


In group II, decreased ER-α expression (ranging from 2.81 to 3.87%, mean 3.31 ± 0.38%) was detected as compared with group I as shown in [Figure 15] and [Figure 16].
Figure 15: A photomicrograph of a section from iliac crest bone of a perimenopausal woman showing decreased estrogen receptor-α expression in fewer positively stained osteoblasts lining bone trabeculae (IHC stain, ×400)

Click here to view
Figure 16: A photomicrograph of a section from iliac crest bone biopsy of a perimenopausal woman showing decreased estrogen receptor-α expression in fewer bone marrow stromal cells (IHC stain, ×400)

Click here to view


In group III, the least ER-α expression (ranging from 1.12 to 2.57%, mean 1.78 ± 0.51%) was observed as shown in [Figure 17].
Figure 17: A photomicrograph of a section from iliac crest bone of a postmenopausal woman showing the least estrogen receptor-α expression in the smallest number of osteoblasts (IHC stain, ´400)

Click here to view


Comparison of the three groups in VEGF and ER-α expression using the ANOVA test showed a highly statistically significant difference (P < 0.001) between groups in both markers as shown in [Table 4] and [Figure 18].
Figure 18: A bar chart showing a comparison of the three groups in VEGF and ER-α expressions. ER-α, estrogen receptor-α; VEGF, vascular endothelial growth factor

Click here to view
Table 4: Comparison of the three groups in VEGF and ERα expressions

Click here to view


Reconsidering BMD, all patients were further subdivided into 14 normal (46.66%), five osteopenic (16.66%), and 11 osteoporotic (36.66%) patients. Comparison of VEGF and ER-α expressions between these groups using the ANOVA test showed a highly statistically significant difference (P < 0.001) in both as shown in [Table 5] and [Figure 19].
Figure 19: A bar chart showing a comparison of the normal, osteopenic, and osteoporotic women in VEGF and ER-α expressions. ER-α, estrogen receptor-α; VEGF, vascular endothelial growth factor

Click here to view
Table 5: Comparison of the normal, osteopenics, and osteoporotics in VEGF and ER-α expressions

Click here to view


Correlating VEGF and ER-α expressions with clinical and laboratory data of the patients

The expression of both VEGF and ER-α showed a highly statistically significant positive correlation (P < 0.001) with E2 levels. However, expression of both VEGF and ER-α showed a highly statistically significant negative correlation (P < 0.001) with age, duration of menstrual irregularities, and menopause.

Correlation of these with age of menarche, BMI, and other laboratory data indicated a nonsignificant correlation (P > 0.05) as shown in [Table 6] and [Figure 20]a and b.
Figure 20: (a) Positive correlation between VEGF expression percentage and E2 levels (pg/ml). (b) Positive correlation between ER-α expression percentage and E2 levels (pg/ml). E2, estradiol; ER-α, estrogen receptor-α; VEGF, vascular endothelial growth factor

Click here to view
Table 6: Correlation of VEGF and ER-α expressions with clinical and laboratory data

Click here to view


Moreover, VEGF expression showed a highly statistically significant positive correlation with ER-α expression (r = 0.968 and P < 0.001) as shown in [Figure 21].
Figure 21: Positive correlation between VEGF expression and ER-α expression. ER-α, estrogen receptor-α; VEGF, vascular endothelial growth factor

Click here to view


Correlations between VEGF, ER-α expressions, E2 levels, and BMD were all highly statistically significantly positive (P < 0.001) as shown in [Table 7] and [Figure 22].
Table 7: Correlations between VEGF, ERα expressions, and E2 levels with BMD

Click here to view


Correlation of VEGF, ER-α expressions, and E2 levels to histomorphometrical bone parameters indicated highly statistically significant positive correlations (P < 0.001) with BV, TbTh, and ObS. In contrast, there were highly statistically significant negative correlations (P < 0.001) with TS and ES as shown in [Table 8] and [Figure 23]a and b.
Figure 22: Positive correlation between VEGF expression and BMD of lumbar spine. BMD, BMD, bone mineral density; VEGF, vascular endothelial growth factor

Click here to view
Figure 23: (a) Positive correlation between E2 levels (pg/ml) and TbTh (mm). (b) Negative correlation between E2 levels (pg/ml) and ES (%). E2, estradiol; ES, eroded surface; TbTh, trabecular bone thickness

Click here to view
Table 8: Correlations between VEGF, ER-α expressions, E2 levels, and histomorphometrical parameters of bone

Click here to view



  Discussion Top


The incidences of osteoporosis and its potentially devastating sequalae as fractures are increasing and are associated with morbidity, disability, and reduced quality of life. Therefore, prevention and treatment of osteoporosis is of major importance [14] .

Estrogen deficiency leads to an increase in the rate of bone remodeling, with resorption exceeding formation. Undoubtedly, this is considered one of the most important factors for postmenopausal osteoporosis [15] .

Coupling between osteogenesis and angiogenesis was reported to be essential for normal bone formation. It was suggested that impairment of angiogenesis would decrease trabecular bone formation [3] .

VEGF is an angiogenic growth factor that couples angiogenesis to osteogenesis [16] . Future therapeutic use of VEGF in the management of bone metabolic pathology therefore depends on a better understanding of the action of VEGF in the bone environment [17] .

This study was designed to investigate the expression of VEGF in bone of postmenopausal osteoporotic patients, assess its relation with BMD, and evaluate the association between VEGF expression, serum E2 levels, and ER-α expression in order to detect its role in the pathogenic pathway of postmenopausal osteoporosis and the possibility of its therapeutic use.

In this study, most patients were overweight and their mean BMI was within that of the obese category (30-40 kg/m). This finding was not surprising as a high prevalence of overweight in Egyptian women has been documented. This may have played a role in the high total percentage of those with low BMD, represented by 16 of 30 patients (53%). It has been suggested that obesity may have a deleterious effect on bone by increasing adipocyte differentiation and fat accumulation while decreasing osteoblast differentiation and bone formation [18] .

In terms of laboratory data, serum calcium, phosphorous, and alkaline phosphatase were all within normal ranges. This result is consistent with Nordin et al. [19] , who reported that Ca absorption remained constant in postmenopausal women till 75 years of age, when it decreased by 30%. Other studies [20],[21] have also reported that these biochemical markers were not affected in primary osteoporosis.

The mean serum E2 level was within the normal range for age for each of our three groups, with a highly statistically significant difference between them. These results were in agreement with several previous studies [21],[22],[23],[24] . The steady decrease in E2 in the perimenopausal period is because of the depletion of ovarian follicles, whereas in the postmenopausal period, E2 is formed by extragonadal conversion of testosterone [22] .

On examining the plain radiograph of dorsolumbar spines, four osteoporotic patients (40%) of group III showed compression fractures in the dorsal spines. This is in agreement with Jergas [25] , who observed that vertebral fractures were the hallmarks of osteoporosis.

Comparison of the three groups in our study in terms of BMD showed a highly statistically significant difference (P < 0.001). Similar results were reported by Khaled and Omar [21] . It was found that all premenopausal patients had normal BMD. On the other extreme, all postmenopausal patients were osteoporotic. Such findings were almost identical to those of Hassab-Elnaby et al.'s study [24] . In their study, none of the premenopausal women were osteoporotic and eight of nine postmenopausal patients (88.8%) were osteoporotic. Also, it was observed that osteoporosis was mostly recorded in postmenopausal women [26] . The gradual loss of the positive effect of estrogen on bone during the perimenopausal period and after menopause is surely a well-established cause of this deterioration in bone mass [27] .

Comparison of the histomorphometrical bone parameters of the three groups showed highly statistically significant differences in all parameters (P < 0.001). This was more or less in agreement with the results of Hassab-Elnaby et al. [24] , who reported highly statistically significant differences between the premenopausal and the postmenopausal considering BV, TbTh, TS, and ES, but they recorded no significant difference in ObS. These findings indicate the gradual microarchitectural deterioration of bone during perimenopausal and postmenopausal periods, which may be attributed to the steady decline in E2 levels. It is worth mentioning that histological examination of postmenopausal bone sections showed widened BM spaces with abundant fat cells. This was similar to the results of Zhao et al. [28] , who reported that histological analysis of postovariectomized bone sections indicated more fat cells and reduced amount of erythropoietic marrow. A decrease in osteoblast differentiation was found to be accompanied by an increase in adipocyte differentiation as both are derived from a common multipotent mesenchymal stem cell [18] .

Moreover, the highly statistical difference between the three groups in our study in the histomorphometric parameters is in agreement with BMD differences measured by DEXA. This finding indicates that although histomorphometry remains the gold standard technique for determining the state of bone, the noninvasive DEXA, showing high efficacy and accuracy, can be used instead. It was documented that DEXA could also identify the risk of fracture as for approximately every 1 SD falls in BMD, there was a two-fold increase in the risk of fracture [24] .

The immunohistochemical detection of VEGF expression in the bone biopsies showed a highly statistically significant difference between premenopausal, perimenopausal, and postmenopausal women in the VEGF percentage of expression. This was in agreement with Ding et al. [29] , who observed decreased expression of VEGF and its association with bone loss in the removed lumbar vertebrae of ovariectomized experimental animals. They related this decrease in the level of VEGF to the withdrawal of estrogen, which was found to be associated with altered bone microcirculation, leading to local abnormal bone metabolism. Decreased VEGF may be the link through which reduced estrogen causes bone angiogenic defects both after an ovariectomy and after menopause. This explanation was indeed confirmed by the highly significant positive correlation between VEGF expression and both plasma E2 levels and ER-α expression in bone biopsies that was found in our study and in a previous study [30] .

Comparison of the VEGF expression between those with normal BMD, osteopenic, and osteoporotic patients showed highly statistically significant differences. Furthermore, a highly statistically significant positive correlation was found between VEGF and BMD. These results are in agreement with Costa et al. [30] , who reported that serum VEGF levels were lower in osteoporotic than in nonosteoporotic women, and with Zhao et al. [28] , who found that in ovariectomized mice, BMD deterioration was correlated to VEGF expression and the associated decreased bone vascularization. However, our results are not in agreement with Cebi et al. [10] , who reported that serum VEGF levels were higher in osteoporotic than nonosteoporotic women and reported a nonsignificant correlation between BMD and serum VEGF in 44 women. However, several previous in vitro and experimental studies supported our finding and reported that VEGF played an anabolic role in bone both by expansion of the vascular bed and by direct bone formation [17] .

VEGF expression showed a highly statistically significant positive correlation with BV, TbTh, and ObS. However, a highly statistical negative correlation with TS and ES was detected. These findings are in agreement with those of Hiltunen et al. [31] , who found that VEGF gene transfer into femurs of experimental animals significantly increased bone formation parameters such as BV and ObS and decreased bone resorption surface as well. These results could be attributed to what was reported earlier by Athanasopoulos et al. [32] that VEGF increased the activity of osteoblasts both directly by its chemotactic effect on them and indirectly by stimulating endothelial cells to increase their expression of BMP-2 and BMP-4, resulting in osteoblast proliferation and differentiation. Also, it was observed that pretreatment of the osteoblast cultures with exogenous VEGF increased alkaline phosphatase release, upregulated the antiapoptotic gene BCL2 expression, and decreased the rates of programmed cell death [8] .


  Conclusion Top


VEGF expression is decreased in bone of postmenopausal osteoporotic patients and is correlated to BMD. Its release is dependent on E2 and mediated through ER-α. These findings suggest that the bone alterations induced by reduced estrogen in postmenopausal osteoporosis may be partly through decreased VEGF release, which makes it one of the possible targets in the treatment of postmenopausal osteoporosis.


  Acknowledgements Top


Conflicts of interest

None declared.

 
  References Top

1.Lane N In: Firestein GS, Budd RC, Harris ED, Mclnnes IB, Ruddy S, Sergent JS (eds.), Metabolic bone disease. Kelley's Textbook of Rheumatology 8th ed. 2009; Philadelphia, PA: Elsevier; 1579-1599.  Back to cited text no. 1
    
2.Boonen S, Ferrari S, Miller PD, Erikson EF, Sambrook PN, Compston J, et al. Postmenopausal osteoporosis treatment with antiresorptives: Effects of discontinuation or long-term continuation on bone turnover and fracture risk - a perspective. J Bone Miner Res 2012; 27 :963-974.  Back to cited text no. 2
    
3.Araldi E, Schipani E Hypoxia, HIFs and bone development. Bone 2010; 47 :190-196.  Back to cited text no. 3
    
4.Tammela T, Enholm B, Alitalo K, Paavonen K The biology of vascular endothelial growth factors. Cardiovasc Res 2005; 65 :550-563.  Back to cited text no. 4
    
5.Bluteau G, Julien M, Magne D, Mallein-Gerin F, Weiss P, Daculsi GGuicheux J VEGF and VEGF receptors are differentially expressed in chondrocytes. Bone 2007; 40 :568-576.  Back to cited text no. 5
    
6.Pufe T, Claassen H, Scholz-Ahrens KE, Varoga D, Drescher W, Franke AT, et al. Influence of estradiol on vascular endothelial growth factor expression in bone: A study in Gottingen miniature pigs and human osteoblasts. Calcif Tissue Int 2007; 80 :184-191.  Back to cited text no. 6
[PUBMED]    
7.Keramaris NC, Calori GM, Nikolaou VS, Schemitsch EH, Giannoudis PV Fracture vascularity and bone healing: A systematic review of the role of VEGF. Injury 2008; 39 (Suppl 2):S45-S57.  Back to cited text no. 7
    
8.Street J, Lenehan B Vascular endothelial growth factor regulates osteoblast survival - evidence for an autocrine feedback mechanism. J Orthop Surg Res 2009; 4 :1-13.  Back to cited text no. 8
    
9.Yen ML, Su JL, Chien CL, Tseng KW, Yang CY, Chen WF, et al. Diosgenin induces hypoxia-inducible factor-1 activation and angiogenesis through estrogen receptor-related phosphatidylinositol 3-kinase/Akt and p38 mitogen-activated protein kinase pathways in osteoblasts. Mol Pharmacol 2005; 68 :1061-1073.  Back to cited text no. 9
[PUBMED]    
10.Cebi H, Aksahin E, Yuksel HY, Celebi L, Aktekin CM, Hapa O, et al. Plasma vascular endothelial growth factor levels are similar in subjects with and without osteoporosis. Joint Dis Relat Surg 2010; 21 :91-97.  Back to cited text no. 10
    
11.Pufe T, Scholz-Ahrens KE, Franke AT, Petersen W, Mentlein R, Varoga D, et al. The role of vascular endothelial growth factor in glucocorticoid-induced bone loss: Evaluation in a minipig model. Bone 2003; 33 :869-876.  Back to cited text no. 11
[PUBMED]    
12.Giannoudis P, Tzioupis C, Almalki T, Buckley R Fracture healing in osteoporotic fractures: Is it really different? A basic science perspective. Injury 2007; 38 (Suppl 1):S90-S99.  Back to cited text no. 12
    
13.Schmidt PJ, Rubinow DR Sex hormones and mood in the perimenopause. Ann N Y Acad Sci 2009; 1179 :70-85.  Back to cited text no. 13
    
14.Jagtap VR, Ganu JV, Nagane NS BMD and serum intact osteocalcin in postmenopausal osteoporosis women. Ind J Clin Biochem 2011; 26 :70-73.  Back to cited text no. 14
    
15.Martin-Millan M, Almeida M, Ambrogini E, Han L, Zhao H, Weinstein RS, et al. The estrogen receptor-α in osteoclasts mediates the protective effects of estrogens on cancellous but not cortical bone. Mol Endocrinol 2010; 24 :323-334.  Back to cited text no. 15
[PUBMED]    
16.Maes C, Carmeliet G In: Ruhrberg C (ed.) Vascular and nonvascular roles of VEGF in bone development. VEGF in development Austin: Springer; 2008; 79-90.  Back to cited text no. 16
    
17.Maes C, Goossens S, Bartunkova S, Drogat B, Coenegrachts L, Stockmans I, et al. Increased skeletal VEGF enhances β-catenin activity and results in excessively ossified bones. EMBO J 2010; 29 :424-441.  Back to cited text no. 17
[PUBMED]    
18.Cao JJ Effects of obesity on bone metabolism. J Orthop Surg Res 2011; 6 :1-7.  Back to cited text no. 18
    
19.Nordin BEC, Need AG, Morris HA, O'Loughlin PD, Horowitz M Effect of age on calcium absorption in postmenopausal women. Am J Clin Nutr 2004; 80 :998-1002.  Back to cited text no. 19
    
20.Simon LS Osteoporosis. Clin Geriatr Med 2005; 21 :603-629.  Back to cited text no. 20
    
21.Khaled HF, Omar SM Tumor necrosis factor alpha expression in iliac bone biopsy and relation to bone histomorphometry in premenopausal and postmenopausal women. Egypt Rheumatol Rehabil 2008; 35 :377-392.  Back to cited text no. 21
    
22.Gruber CJ, Tschugguel W, Schneeberger C, Huber JC Production and actions of estrogens. N Engl J Med 2002; 346 :340-352.  Back to cited text no. 22
    
23.Grady D Management of menopausal symptoms. N Engl J Med 2006; 355 :2338-2347.  Back to cited text no. 23
    
24.Hassab-Elnaby MM, El-Ganzouri AM, Abdel-Moneeim HF, Moussa MH, Abaza NM Osteoprotegerin (OPG) and histomorphometric assessment in human bone in relation to menopausal status 2007; 85-113.  Back to cited text no. 24
    
25.Jergas M In: Grampp S (ed.) Radiology of osteoporosis. Medical radiology: Diagnostic imaging 2 nd ed. Heidelberg: Springer; 2008; 77-104.  Back to cited text no. 25
    
26.Li S, He H, Ding M, He C The correlation of osteoporosis to clinical features: A study of 4382 female cases of a hospital cohort with musculoskeletal symptoms in southwest China. BMC Musculoskeletal Disorders 2010; 11 :1-9.  Back to cited text no. 26
    
27.Estok PJ, Sedlak CA, Doheny MO, Hall R Structural model for osteoporosis preventing behavior in postmenopausal women. Nurs Res 2007; 56 :148-158.  Back to cited text no. 27
    
28.Zhao Q, Shen X, Zhang W, Zhu G, Qi J, Deng L Mice with increased angiogenesis and osteogenesis due to conditional activation of HIF pathway in osteoblasts are protected from ovariectomy induced bone loss. Bone 2012; 50 :763-770.  Back to cited text no. 28
    
29.Ding WG, Wei ZX, Liu JB Reduced local blood supply to the tibial metaphysis is associated with ovariectomy-induced osteoporosis in mice. Connect Tissue Res 2011; 52 :25-29.  Back to cited text no. 29
    
30.Costa N, Paramanathan S, Mac Donald D, Wierzbicki AS, Hampson G Factors regulating circulating vascular endothelial growth factor (VEGF): Association with bone mineral density (BMD) in post-menopausal osteoporosis. Cytokine 2009; 46 :376-381.  Back to cited text no. 30
    
31.Hiltunen MO, Ruuskanen M, Huuskonen J, Mahanen AJ, Ahonen M, Rutanen J, et al. Adenovirus-mediated VEGF-A gene transfer induces bone formation in vivo. FASEB J 2003; 17 :1147-1149.  Back to cited text no. 31
    
32.Athanasopoulos AN, Schneider D, Keiper T, Alt V, Pendurthi UR, Liegibel UM, et al. Vascular endothelial growth factor (VEGF)-induced upregulation of CCN1 in osteoblasts mediates proangiogenic activities in endothelial cells and promotes fracture healing. J Biol Chem 2007; 282 :26746-26753.  Back to cited text no. 32
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18], [Figure 19], [Figure 20], [Figure 21], [Figure 22], [Figure 23]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]


This article has been cited by
1 A three-dimensional block structure consisting exclusively of carbon nanotubes serving as bone regeneration scaffold and as bone defect filler
Manabu Tanaka,Yoshinori Sato,Hisao Haniu,Hiroki Nomura,Shinsuke Kobayashi,Seiji Takanashi,Masanori Okamoto,Takashi Takizawa,Kaoru Aoki,Yuki Usui,Ayumu Oishi,Hiroyuki Kato,Naoto Saito,Syam Nukavarapu
PLOS ONE. 2017; 12(2): e0172601
[Pubmed] | [DOI]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Aim of the work
Patients and methods
Results
Discussion
Conclusion
Acknowledgements
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed1251    
    Printed17    
    Emailed0    
    PDF Downloaded108    
    Comments [Add]    
    Cited by others 1    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]