Rapamycin inhibits the proliferation of endothelial cells in hemangioma by blocking the mTOR-FABP4 pathway.

FABP4 is widely expressed in both normal and pathologic tissues. It promotes cell proliferation, survival and migration of endothelial cells, and therefore, angiogenesis. However, the role of FABP4 in hemangioma or hemangioma endothelial cells (HemECs) has not been explored. In this study, we investigated whether FABP4 directly regulates the proliferation of HemECs. The expression of cell cycle checkpoint genes was analyzed with the microarray data of human dermal microvascular endothelial cells (HDVECs) and infantile hemangioma endothelial cells. Real-time RT-PCR and western blotting were used to examine the expression of FABP4 in HemECs. Next, the FABP4 expression was inhibited in HemECs using siRNA or rapamycin and upregulated using retroviral transduction of HemECs to assess its influence on proliferation of HemECs. The microarray data showed that cell cycle checkpoint genes were upregulated in HemECs. Moreover, HemECs showed significantly higher proliferation rates than HDVECs. The expression of FABP4 and mTOR was increased in the HemECs. While FABP4 knockdown reduced the BrdU incorporation and cell number of HemECs as expected, cell proliferation was accelerated by FABP4 over-expression. Moreover, rapamycin (10nM) inhibited mTOR-FABP4 signaling and HemEC proliferation. Taken together, these results indicated that mTOR signaling pathway-activated FABP4 directly regulates the proliferation of endothelial cells in hemangioma. Rapamycin and inhibitors of FABP4 have therapeutic potential for treating infantile hemangiomas.

Mast cells in infantile haemangioma possess a primitive myeloid phenotype.

AIMS: Recent reports on infantile haemangioma (IH) have demonstrated a primitive population of interstitial cells expressing the embryonic transcription factor, Nanog, with decreasing abundance during involution. In this report we investigated the expression of Nanog on mast cells in all three phases of IH progression. METHODS: Paraffin-embedded sections of six proliferating, six involuting and six involuted IH lesions were used to investigate the expression of tryptase, Nanog, CD45, CD34 and GLUT-1 by immunostaining. RESULTS: Mast cells, identified by their expression of tryptase, were located in the interstitium of IH lesions. 93%, 42% and 0% of these tryptase(+) cells also expressed Nanog, in proliferating, involuting and involuted IH, respectively. CONCLUSIONS: The identification of an abundant population of tryptase(+)/Nanog(+) cells in IH is novel. The relative loss of Nanog expression as IH involutes may be a result of maturation and/or proliferation of these cells. This report supports the primitive nature of IH.

Endothelial nitric oxide synthase expression in pulmonary capillary hemangiomatosis.

Pulmonary capillary hemangiomatosis (PCH) is an unusual disorder characterized by the proliferation of capillaries in the alveolar septa and pulmonary interstitium. Originally conceived as a primary idiopathic disorder of the pulmonary microcirculation, recent studies have demonstrated that PCH may be associated with other pathologies. Nitric oxide (NO) is a gaseous free radical with protean biological effects that is released during the intracellular conversion of arginine to citrulline. Nitric oxide synthases (NOS) mediate the production of NO and the release of NO in the microvasculature is specifically catalyzed by endothelial NOS (NOS-III). As NOS contributes to angiogenesis and is reduced in the hypertensive pulmonary microcirculation, we examined the expression of NOS-III protein in situ in the lungs of patients with PCH. Reduced microvascular expression of NOS-III protein by endothelial cells was observed in 4/6 (67%) cases of PCH, and all of these showed concomitant pulmonary vascular hypertensive remodeling. In 2/6 (33%) cases of PCH with no morphologic evidence of pulmonary hypertensive arteriopathy, endothelial expression of NOS-III protein was judged to be either minimally reduced or normal. These findings suggest that NOS-III is specifically reduced in PCH when pulmonary arterial hypertensive remodeling is concomitantly present.

Nitric oxide synthases in Kaposi's sarcoma are expressed predominantly by vessels and tissue macrophages.

Kaposi's sarcoma (KS) is a tumor of presumed vascular origin frequently found in patients with AIDS. Recent data suggest that the development of KS is linked with the presence of a newly recognized herpesvirus, human herpesvirus type 8. Nitric oxide (NO), a messenger molecule with vasoactive, antitumor, and antimicrobial effects, is produced by three isoforms of nitric oxide synthases (NOS). In the present report, we investigated the expression of NOS isoforms in KS. By NADPH-diaphorase histochemistry, NOS activity was detectable in endothelia and CD45+ cells within KS lesions. Reactivity for endothelial NOS (eNOS) was found in blood vessel endothelia; however, eNOS reactivity was negative in KS spindle cells in 12 of 17 tumors, and moderately positive in the other 5 lesions. In contrast to KS, tumor cells in three hemangiomas and one angiosarcoma were strongly positive for eNOS. Inducible NOS (iNOS) was absent from KS tumor cells but was found regularly in CD45+, HLA-DR+ cells within the lesions. In five KS-derived spindle cell cultures, neither eNOS nor iNOS proteins were detectable. The sporadic expression of eNOS by KS spindle cells in vivo and the absence of eNOS protein from KS spindle cells in tissue cultures argue against the possibility that the cells are derived from blood vessel endothelia. The consistent expression of iNOS by CD45+, HLA-DR+ cells within KS lesions strongly suggests that leukocyte-derived NO participates in the pathology of this tumor.

Inducible nitric oxide synthase is expressed in granuloma pyogenicum.

It is known that granuloma pyogenicum (GP) grows rapidly but cherry angioma (CA) does not, although they show the same histological features of hyperplasia of the capillary vessels. Although some cytokines have been thought to be angiogenic factors, a nitric oxide (NO) synthase-dependent effector mechanism is reported to be involved in macrophage-derived angiogenesis in humans. Under the hypothesis that this mechanism is also involved in the more rapid growth of GP, we examined the expression of inducible NO synthase (iNOS) in GP as compared with that in CA. The expression of iNOS mRNA was detected in GP, but not in CA, by reverse transcription-polymerase chain reaction (RT-PCR) analysis. In situ hybridization (ISH) demonstrated iNOS mRNA expression in endothelial cells and to a lesser extent round cells and spindle cells in the myxomatous stroma in GP. Immunohistochemical study showed that iNOS protein was expressed in endothelial cells, infiltrating round cells and spindle cells in the stroma in GP. These results suggested that a NOS-dependent mechanism may be involved in angiogenesis and the rapid growth of GP.

Strong expression of kinase insert domain-containing receptor, a vascular permeability factor/vascular endothelial growth factor receptor in AIDS-associated Kaposi's sarcoma and cutaneous angiosarcoma.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), plays an important role in the angiogenesis associated with the growth of many human and animal tumors. VPF/VEGF stimulates endothelial cell growth and increases microvascular permeability by interacting with two endothelial cell tyrosine kinase receptors, KDR and flt-1. We studied 16 cases of AIDS-associated Kaposi's sarcoma (KS), 2 cases of cutaneous angiosarcoma, and 6 cases of capillary hemangioma by in situ hybridization for expression of VPF/VEGF, KDR, and flt-1 mRNAs. We also performed immunohistochemical staining for VPF/VEGF protein in 15 cases. Tumor cells in KS and angiosarcoma strongly expressed KDR but not flt-1 mRNA. Endothelial cells in small stromal vessels in and around these tumors strongly expressed both KDR and flt-1 mRNAs. Tumor cells expressed VPF/VEGF mRNA strongly in only one case of KS, adjacent to an area of necrosis. This was also the only case in which the tumor cells stained substantially for VPF/VEGF protein. VPF/VEGF mRNA and protein were, however, strongly expressed by squamous epithelium in areas of hyperplasia and near areas of ulceration overlying tumors. VPF/VEGF mRNA was also expressed focally at lower levels by infiltrating inflammatory cells, probably macrophages. The strong expression of both KDR and flt-1 in small stromal vessels in and around tumors suggests that VPF/VEGF may be an important regulator of the edema and angiogenesis seen in these tumors. The strong expression of KDR by tumor cells in KS and angiosarcoma implies that VPF/VEGF may also have a direct effect on tumor cells. Tumor cells in four of six capillary hemangiomas strongly expressed both KDR and flt-1 mRNAs in contrast to the high level expression of only KDR observed in the malignant vascular tumors studied. Neither VPF/VEGF mRNA or protein were strongly expressed in capillary hemangiomas. VPF/VEGF and its receptors may play an important but as yet incompletely understood role in the pathogenesis of both benign and malignant vascular tumors.

Histochemical study on hemangiomas in childhood.

The study was carried out on a material obtained by a biopsy from 18 children having different types of hemangiomas (capillary, cavernous and combined). Histochemical reactions were applied for the following enzymes: Succenatedehydrogenase (SDH), lactatdehydrogenase (LDH), cytochromoxidase, acid phosphatase, alkaline phosphatase and paraspecific esterases. Differently reduced activity of the succenatedehydrogenase and the cytochromoxidase in the endothelial cells and the pericytes was established. A reduced enzyme activity was observed also in a large part of the cases of acid phosphatase. The activity of the alkaline phosphatase and paraspecific esterases was greatly increased. Based on their observations the authors relate the hemangiomas to the tumour formations with a good prognosis.