Carcinosarcomas of the female genital tract are a heterogeneous group of aggressive malignant neoplasms characterized by poor prognosis that contain elements expressing both carcinomatous and sarcomatous characteristics. In this study specimens from 25 patients were treated with labeled antibodies to vascular endothelium (FVIII), and to vascular endothelial growth factor (VEGF) for analysis of angiogenesis, and to vascular endothelial growth factor receptor 3 (VEGFR-3) for analysis of lymphangiogenesis, in 11,099 vessels. Automated quantitative image analysis was used and the results were compared with clinical data. Microvessel density increased from a median value of 18.32 vessels/mm(2) in non-neoplastic stroma to 131.25 vessels/mm(2) in neoplasms. In areas around tumor islets expressing predominantly epithelial carcinomatous characteristics, microvessel density was increased three-fold compared with the islets themselves. Vessels were arranged in a garland-type pattern, or in bursts, and they exhibited directional angiogenesis. Clinical indicators of poor survival were high tumor stage (p=0.002) and age above 65 (p=0.0769). A high number of small vessels (16-300 mum(2) in cross-sectional area) predicted poor survival (p=0.0149), and more so in tumors exhibiting predominantly sarcomatous characteristics (p=0.0087). Tumor tissue area above the median exhibiting VEGF expression was also a sign of poor survival (p=0.0267), as was an area of positive staining for VEGFR-3 exceeding the median (p=0.00487). In this study, active angiogenesis (increased number of vessels, variable in shape and exhibiting decreased antibody staining intensity) was a distinct feature of carcinosarcomas, its extent and distribution depending upon neoplasm morphology. Increased vessel numbers and increased VEGF and VEGFR-3 expression indicated poor survival.
Angiogenesis is an essential biological process not only in embryogenesis, but also in the progression of several major diseases, including cancer, diabetes, and inflammation. Excessive vascularization can also contribute to some cardiovascular pathologies, such as atherosclerosis, but contradictory reports still prevail regarding its impact on aortic stenosis. Using immunohistochemical techniques, we assessed the vascular density and distribution of angiogenesis (FVIII) and vascular endothelial growth factor (VEGF) expression as well as the expression of 2 VEGF receptors, Flt-1 and Flk-1, in 55 nonrheumatic and 6 control aortic valves. In the light of the fact that the angiogenic effect of VEGF is mediated by sustained formation of nitric oxide, the samples were also immunostained with 3 nitric oxide synthase (eNOS, iNOS, and nNOS) antibodies. The immunohistochemical findings of VEGF and its receptors were verified by immunoblotting techniques. Vascular density was highest in the cases with moderate valve stenosis, and the mean number of FVIII-positive blood vessels was 1.7 +/- 1.9 vessels/mm(2) in the diseased valves, whereas the normal valves contained no blood vessels. Vascular density was significantly higher in the cases showing chronic inflammation (P = 0.007). Interestingly, the patients receiving statin therapy had significantly lower vascular densities than those not receiving such therapy (P = 0.001). Diseased valves showed distinct VEGF, Flt-1, Flk-1, and eNOS positivity of activated endothelial, stromal fusiform myofibroblastic, and histocytic cells. In contrast, immunoreactivity for iNOS and nNOS was seen only in nonendothelial stromal cells, and their expression was weaker. Enhanced vascular density was significantly associated with increased expression of Flk-1 (P = 0.028 for endothelial and P = 0.009 for stromal cells) and with endothelial eNOS expression (P = 0.024). A similar tendency was also observed for VEGF, but not for Flt-1. Our results show a distinct angiogenic response and the presence of angiogenic factors in nonrheumatic aortic valve stenosis, suggesting that angiogenesis may influence on the evolution of this disease.
BACKGROUND AND AIM OF THE STUDY: Aortic valve stenosis (AS) is an actively regulated pathobiological process that shows some hallmarks of atherosclerosis. Apelin and its receptor, APJ, are highly expressed in the heart, and the proposed effects of the apelin-APJ system are opposite to those of the angiotensin II-AT1-receptor pathway. The role of the apelin-APJ signaling pathway in calcified aortic valve disease is unknown. METHODS: The study involved the characterization and comparison of expression of apelin and APJ as well as angiotensin II receptors (AT1 and AT2) in the aortic valves of patients with normal valves (n = 6), aortic regurgitation (n = 9 AR), regurgitation and fibrosis/mild sclerosis (n = 14), and AS (n = 25). RESULTS: By employing the reverse-transcriptase polymerase chain reaction (RT-PCR), the gene expression of apelin (3.63-fold, p = 0.001) and the APJ receptor (2.70-fold, p = 0.01) were shown to be significantly up-regulated in stenotic valves when compared to controls. In addition, APJ receptor mRNA levels were higher (2.9-fold, p = 0.010) in the AR + sclerosis group when compared to controls. Using immunohistochemistry, apelin was shown to be localized in stenotic aortic valves to the valvular endothelial layer of the aortic valve, to vascular endothelial cells in neovessels, and to fibroblasts and macrophages adjacent to vessels in the stromal area. AT2-receptor mRNA levels were 90% (p
OBJECTIVE: Appropriate clinical management of cases of FIGO Grade I and II endometrial carcinoma relies heavily on the determination of myometrial invasion (MI). There are no reports addressing expression of the cell adhesion molecule CD44 in the subset of Grade I and II endometrioid carcinoma (EC) as it relates to prognosis, including MI. METHODS: Immunohistochemical staining for CD44s and CD44v6 was evaluated in 40 hysterectomy specimens with Grade I and II EC, including 11 noninvasive ECs, 14 with MI 50%). Staining characteristics according to the presence of MI and vascular space invasion (VSI) were evaluated. Strong membranous staining of >10% of tumor cells was interpreted as positive. RESULTS: CD44v6 staining was positive in 20% (8/40) of cases, including 45% (5/11) of EC without MI but only 10% (3/29) with MI (P = 0.025). CD44v6 staining was not present in deeply invasive tumors (0/15), while it was present in 8/25 superficially or noninvasive tumors (P = 0.016). Sensitivity and specificity were 25 and 100%, respectively, using CD44v6 in evaluating deep myometrial invasion. CD44s showed a trend toward positive staining when comparing noninvasive versus invasive tumors and noninvasive/superficially invasive versus deeply invasive tumors (P = 0.08 and 0.12, respectively). CD44s or CD44v6 staining was highly specific for absence of VSI, although statistical comparison did not reach significance. CONCLUSION: Deeply invasive EC was associated with a consistent lack of CD44v6 expression. This may have potential clinical utility if this finding is demonstrated in further study of prehysterectomy sampling specimens containing EC.
In this study we investigated the number of blood vessels and vascular proliferation in subepithelial areas of 80 cervical condylomas and cervical intraepithelial neoplasias (CIN). The number of blood vessels was determined by counting factor VIIIRAg-positive vascular channels in areas beneath the epithelial lesions. Vascular proliferation was evaluated by determining the number of proliferating cell nuclear antigen (PCNA)-positive endothelial cells in the subepithelial connective tissues. The results were compared with the expression of human papillomavirus (HPV) DNA subgroups (6/11 (low-risk) and 16/18/31/33/35 (high-risk) of the lesions, as determined by dot-blot and in situ hybridization, and with epithelial cell proliferation as determined by immunohistochemistry for PCNA. Also p53 immunohistochemistry of the lesions was performed. Even though CIN II-III lesions on average contained more factor VIIIRAg-positive blood vessels compared to condylomas and CIN I lesions, no significant association was found between their number and the degree of dysplasia. However, moderate or strong PCNA staining in vascular endothelial cells was seen significantly more often in CIN II-III lesions than in condylomas and CIN I lesions (p = 0.008): 34/80 (45%) cases contained detectable HPV DNA as determined by dot-blot or in situ hybridization. There was no correlation between the presence or absence of HPV DNA and the number of PCNA-positive endothelial cells. Nine cases showed p53-positive cell nuclei and in three cases there was more than 1% positive nuclei in the lesion. No association was found between the vascularity or the number of PCNA-positive endothelial cells and the p53 immunoreactivity. The increased proliferative activity of endothelial cells in CIN II-III lesions suggests that they are angiogenically more active than condylomas and CIN I lesions. This activity does not, however, depend on the HPV or p53 status. This is the first report in which endothelial cell PCNA positivity was used as a marker for vascular proliferation.
Increased vascularization is considered an important contributing factor for plaque vulnerability. Microvascular proliferative disease in patients with diabetes results in renal damage and visual loss. We assessed the hypothesis that vascularization in carotid atherosclerotic tissue is increased in diabetic patients, especially in the critical shoulder regions of the plaque.
Carotid endarterectomy specimens, clinical data, and blood samples were collected from patients with symptomatic carotid artery stenosis (median 85 days after clinical event) and pharmacologic treatment for diabetes (n = 26) or no diabetes (n = 85). Plaques were fixed in formalin and transverse tissue sections prepared. Histopathology and immunohistochemistry were performed for detection of endothelial cells (anti-CD34), macrophages (anti-CD68), vascular endothelial growth factor (VEGF), and its receptor (VEGFR-2). Neovascularization was assessed as CD34(+) neovessel density in the entire section area and by the presence or absence of CD34(+) vessels in the shoulder and cap regions of the plaques.
The patient groups did not differ significantly in neovascularization in the entire transverse sections (2.0 vs 2.1 vessels/mm(2); P = .61) or in the fibrous cap (52% of the patients in both groups; P = .95). Neovascularization of the plaque shoulder regions was observed in 52% of the diabetic patients and in 26% of the nondiabetic patients (P = .028). VEGF-stained areas were similar in the two patient groups (0.4% and 0.2% of shoulder area; P = .61). Patients with diabetes had more VEGFR-2 (1.0% vs 0.2% of shoulder area; P 90 days, 50% of patients; P = .002), independently of diabetes status.
Diabetes was associated with increased vascularization of the shoulder regions in patients with symptomatic carotid atherosclerotic plaques. This was accompanied by increased expression of VEGFR-2. The increased vascularization of the plaque shoulder regions may help explain why patients with diabetes are at increased risk of atherosclerotic complications.
Bronchiolitis obliterans organizing pneumonia (BOOP) and usual interstitial pneumonia (UIP; ie, cryptogenic fibrosing alveolitis of mural type, CFA) are clinically and histologically distinguishable interstitial lung diseases. Both contain intraluminal lesions of newly formed fibromyxoid connective tissue. In BOOP, the fibromyxoid lesions are susceptible to complete reversal, but in UIP they are supposed to participate in the remodeling of the interstitium. Our hypothesis was that capillarization of the intraluminal fibromyxoid lesions is more frequent in BOOP compared with UIP. In this study, we stained diagnostic thoracoscopic or open lung biopsy specimens of patients with BOOP (n = 9) and UIP (n = 10) with antibodies against human laminin, von Willebrand factor, and CD34 to reveal the microvasculature of intraluminal fibromyxoid lesions. Our results show that in BOOP there is abundant capillarization in the newly formed intraluminal fibromyxoid lesions often reminiscent of granulation tissue. The mean number of capillaries per area unit (mm2) was 107 +/- SD 74 in samples stained for laminin, 103 +/- SD 46 for von Willebrand factor, and 63 +/- SD 36 for CD34. In marked contrast, in UIP, the corresponding accounts were significantly lower, being 14 +/- SD 15 for laminin (P
There is increasing evidence that renin-angiotensin system (RAS) may play a major role in the actively regulated fibrocalcific process in aortic valve stenosis (AS), but the gene expression or function of (pro)renin receptor ((P)RR), prorenin and renin or angiotensin converting enzyme 2(ACE2)/angiotensin-(1-7)/Mas receptor axis in calcific aortic valve disease is not known.
We characterized expression of (P)RR, ACE2 and Mas receptor as well as renin, prorenin and angiotensin II type 2 (AT(2)) receptors in human aortic valves, and compared normal control valves (n = 11) with valves obtained from patients with aortic regurgitation (AR, n = 14), AR with fibrosis (n = 20) and AS (n = 61). By immunohistochemistry (P)RR positive staining was seen in the valvular endothelial cells of control and in the neovessels of stenotic valves. By RT-PCR, renin mRNA levels were 72% (P = 0.001) and prorenin mRNA levels 64% lower (P = 0.002) in stenotic aortic valves compared to control valves. ACE2, Mas receptor and AT(2)-receptor mRNA levels were 69% (P
Angiogenesis is a key process in tumour growth and metastasis, and Factor-VIII microvascular density has been found to influence prognosis among endometrial carcinoma patients. The CD105/endoglin antibody has been reported to preferentially bind to activated endothelial cells in tissues participating in angiogenesis, and we therefore wanted to compare the prognostic significance of CD105/endoglin to that of Factor-VIII. In a population-based endometrial carcinoma study with long (median 11.5 years) and complete patient follow-up, mean intratumour microvascular density (MVD) assessed using CD105/endoglin was investigated and compared with previous data for MVD assessed using Factor-VIII. MVD by CD105/endoglin was significantly correlated with MVD by Factor-VIII (p=0.001). However, tumours within the two groups defined by the upper and lower quartiles for CD105/endoglin-MVD were both significantly more often metastatic (FIGO-stage III/IV; p=0.03), with high tumour cell proliferation by Ki67 (p=0.007) and with reduced survival (p=0.036) as compared with the intermediate groups. In Cox regression analysis, CD105/endoglin-MVD showed independent prognostic influence when analysed together with patient age, FIGO stage, histologic subtype, histologic grade and Factor-VIII-MVD, while the latter lost its prognostic impact when CD105/endoglin was included. In the subgroup with high MVD, there was a tendency towards improved response to radiation therapy. In conclusion, CD105/endoglin-MVD is significantly associated with FIGO stage, tumour proliferation and prognosis in endometrial carcinoma, indicating that this is a better angiogenic marker in these tumours.
Increased angiogenesis and expression of antibodies to vascular endothelial growth factor (VEGF), an angiogenic agent, have been shown in the tumor development of many tissues. Areas of skin expressing VEGF and total volume of vessels expressing laminin in the wall were measured in chemical carcinogen-exposed mice using CAS-200 morphometry apparatus having a sensitivity exceeding 99% and reproducibility exceeding 99%. The area of VEGF expression was increased in carcinogen-exposed skin, dysplasia and in well-differentiated squamous cell carcinomas, but decreased in squamous cell carcinomas with decreased degree of differentiation. The vessel volume increased prior to the formation of tumors in carcinogen-exposed skin as well as in highly malignant neoplasms. In well-differentiated squamous cell carcinomas with an expansive growth pattern, the vessels were parallel to the basal membrane, in moderately differentiated tumors the vessels were in the direction of tumor invasion, and in poorly differentiated tumors, active angiogenesis consisted of numerous, enlarged vessels within the tumor. This study showed increased VEGF expression and number of vessels occurring in early stages of skin tumor development, pointing to a role of angiogenesis in chemical risk assessment and in cancer prevention. Altered vessel structure and vessel arrangement were distinct in later stages of tumor growth and in malignant neoplasms, pointing to the utility of detailed vessel analysis in neoplasm characterization.