Outcome after injections of crushed tablets in intravenous drug abusers in the Helsinki University Central Hospital.

OBJECTIVE: To retrospectively analyse injection drug users (IDUs) with complications after intra- or extra-vasal administration of dissolved tablets. DESIGN: A retrospective study. METHODS: The hospital discharge registers were used to identify the patients admitted in different clinics in Helsinki University Central Hospital during 2000-2005. The patient demographics and social background were clarified. The type of the crushed drugs, the injection route and the timing of administration were registered. Medical interventions, examinations and surgical procedures were recorded. RESULTS: Between January 2000 and December 2005, 24 patients had been treated on 30 occasions for manifestations caused by injecting crushed tablets. The main types of manifestations were acute limb ischaemia (16 patients) and infection (eight patients), and eight cases led to distal or proximal amputations. Men (19 of 24) were affected more frequently than were women (5 of 24). Their ages ranged between 20 and 39 years (mean: 26 years). All the patients had a previous history of intravenous drug abuse, and they lived in Greater Helsinki region. The incidence of seropositivity for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) was 33% (n=8), 88% (n=21) and 4% (n=1), respectively. The time between injection and presentation to the Emergency Department varied between 3h and 10 days (mean: 62 h). Buprenorphine was the most commonly used drug in 10 of the 24 patients, and benzodiazepine derivatives were also used in 11 of the 24 patients. CONCLUSIONS: Intra- or extra-vasal administration of dissolved tablets leads to serious consequences, including limb amputations. Vascular and soft-tissue imaging may be helpful in the diagnosis. Prompt drainage of any abscess and fasciotomies for compartment syndrome treatment are essential. Controversy exists over the best medical therapy.

VEGFR-3 in adult angiogenesis.

Vascular endothelial growth factor receptor 3 (VEGFR-3, Flt-4), the receptor for vascular endothelial growth factors (VEGFs) C and D, is expressed on lymphatic endothelium and may play a role in lymphangiogenesis. In embryonic life, VEGFR-3 is essential for blood vessel development. The purpose of this study was to investigate whether VEGFR-3 is also involved in blood vessel angiogenesis in the adult. This was studied in human tissues showing angiogenesis and in a model of VEGF-A-induced iris neovascularization in the monkey eye, by the use of immunohistochemistry at the light and electron microscopic level. VEGFR-3 was expressed on endothelium of proliferating blood vessels in tumours. In granulation tissue, staining was observed in the proliferative superficial zone in plump blood vessel sprouts, in the intermediate zone in blood vessels and long lymphatic sprouts, and in the deeper fibrous zone in large lymphatics, in a pattern demonstrating that lymphangiogenesis follows behind blood vessel angiogenesis in granulation tissue formation. At the ultrastructural level, VEGFR-3 was localized in the cytoplasm and on the cell membrane of endothelial cells of sprouting blood vessels and sprouting lymphatics. In monkey eyes injected with VEGF-A, blood vessel sprouts on the anterior iris surface and pre-existing blood vessels in the iris expressed VEGFR-3. In conclusion, these results support a role for VEGFR-3 and its ligands VEGF-C and/or VEGF-D in cell-to-cell signalling in adult blood vessel angiogenesis. The expression of VEGFR-3 in VEGF-A-induced iris neovascularization and in pre-existing blood vessels exposed to VEGF-A suggests that this receptor and possibly its ligands are recruited in VEGF-A-driven angiogenesis.

Lymphatic versus blood vascular endothelial growth factors and receptors in humans.

Three different growth factor systems have been described acting via endothelial cell-specific receptor tyrosine kinases (RTKs). These are vascular endothelial growth factors (VEGFs), angiopoietins, and ephrins. Recent studies on gene targeting suggest that they play critical roles in embryonic development and contribute to the integrity and responses to environmental factors in the adult vasculature. Coagulation, inflammation, immune response regulation, vascular tone, stromal component synthesis, and angiogenesis are all dependent on the physiological and pathological events that affect endothelial cells in the heart, arteries, veins, and lymphatic vessels. Angiogenesis, the formation of new blood vessels from preexisting ones, takes place in adults only during hormonal control of female reproduction. All other activation of angiogenesis in adulthood occurs in response to injury or pathological processes such as tumorigenesis, diabetes, or inflammatory conditions. Insufficient growth of collateral vessels is a major problem in atherosclerotic cardiovascular disease. Controlled stimulation of angiogenesis would be of therapeutic value. Lymphangiogenesis, the mechanisms involved in the development of lymphatic vessels, was studied intensively nearly a century ago, although since then it has been neglected, perhaps because, unlike the disorders of blood vessels, those of the lymphatic vessels are seldom life-threatening. Interrupting this one-way system can cause severe disorders, including liver dysfunction, genetic disease (e.g., Milroys disease), and degenerative disease (e.g., primary lymphangiosclerosis). Recently, novel growth factors, receptors, cell surface proteins, and transcription factors have been found which play a role in the lymphatic endothelium. These are VEGF-C, VEGF-D, VEGFR-3, LYVE-1, podoplanin, and Prox-1. Until recently lymphatic vessels have been difficult to study due to a lack of appropriate tools. Monoclonal antibodies raised against VEGFR-3 and against its ligands, VEGF-C and VEGF-D, have offered an insight into expression studies in tissues. In this review, we summarize the recent data on VEGFs in the human vasculature.

VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissues.

Recently, vascular endothelial growth factor receptor 3 (VEGFR-3) has been shown to provide a specific marker for lymphatic endothelia in certain human tissues. In this study, we have investigated the expression of VEGFR-3 and its ligands VEGF-C and VEGF-D in fetal and adult tissues. VEGFR-3 was consistently detected in the endothelium of lymphatic vessels such as the thoracic duct, but fenestrated capillaries of several organs including the bone marrow, splenic and hepatic sinusoids, kidney glomeruli and endocrine glands also expressed this receptor. VEGF-C and VEGF-D, which bind both VEGFR-2 and VEGFR-3 were expressed in vascular smooth muscle cells. In addition, intense cytoplasmic staining for VEGF-C was observed in neuroendocrine cells such as the alpha cells of the islets of Langerhans, prolactin secreting cells of the anterior pituitary, adrenal medullary cells, and dispersed neuroendocrine cells of the gastrointestinal tract. VEGF-D was observed in the innermost zone of the adrenal cortex and in certain dispersed neuroendocrine cells. These results suggest that VEGF-C and VEGF-D have a paracrine function and perhaps a role in peptide release from secretory granules of certain neuroendocrine cells to surrounding capillaries.

Vascular endothelial growth factor-C and its receptor VEGFR-3 in the nasal mucosa and in nasopharyngeal tumors.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are important regulators of blood and lymphatic vessel growth and vascular permeability. Both blood and lymphatic vessels of the upper respiratory tract play important roles in pathological conditions, such as infections and tumors. Here we have studied the expression of VEGF-C and its receptor VEGFR-3 in the upper respiratory system by Northern blot analysis and immunohistochemistry of human tissues, and in situ mRNA hybridization of developing mouse embryos and beta-galactosidase staining of mouse embryos having a LacZ marker gene in the VEGFR-3 gene locus. The results demonstrate expression of VEGF-C and VEGFR-3 in the developing and adult nasal respiratory epithelium and in the nasal vascular plexus, respectively. Unlike in most other tissues, in the nasal mucosa VEGFR-3 is expressed in both blood and lymphatic vessels. Expression of VEGF-C was also detected in nasal and nasopharyngeal tumor islands, which were surrounded by VEGFR-3-positive angiogenic blood vessels. These results suggest that VEGF-C and VEGFR-3 have a role in the development of the nasal submucosal vascular plexus and in its normal function and that they are associated with angiogenesis in nasal and nasopharyngeal tumors.

Lack of lymphatic vascular specificity of vascular endothelial growth factor receptor 3 in 185 vascular tumors.

BACKGROUND: Among the molecules important to angiogenesis and lymphangiogenesis is vascular endothelial growth factor receptor 3 (VEGFR-3), a member of the receptor tyrosine kinases of endothelial cells. This receptor is expressed consistently in normal lymphatics, lymphangiomas, and in Kaposi sarcoma, but data regarding other vascular tumors are scant. METHODS: In this study the authors immunohistochemically examined VEGFR-3 expression in 82 benign, 31 borderline, and 72 malignant vascular tumors using a monoclonal antibody to VEGFR-3, heat-induced epitope retrieval, and an avidin-biotin-peroxidase detection system. RESULTS: Although normal mesenchymal tissues showed VEGFR-3 only in the lymphatics, benign and malignant vascular tumors and neovascularization of nonendothelial tumors showed widespread VEGFR-3 distribution. All lymphangiomas and Kaposi sarcomas showed consistent VEGFR-3 reactivity. Among the hemangiomas, spindle cell hemangiomas and 80% of capillary (including all lobular capillary hemangiomas) were positive whereas the endothelium of cavernous, venous, and epitheloid hemangiomas were positive in a minority of cases (20%, 27%, and 33%, respectively). Among the borderline lesions, Kaposiform hemangioendotheliomas were intensely positive whereas epithelioid hemangioendotheliomas were positive in 11 of 29 cases (38%). Angiosarcomas showed VEGRF-3 reactivity in the majority of cases (48 of 60 cases; 80%). The nonepithelioid variants more often were positive (40 of 45 cases; 89%) than the epithelioid variants, of which 8 of 15 (53%) showed positive tumor cells. Nonvascular tumors (including perivascular tumors, other sarcomas, melanomas, carcinomas, and large cell lymphomas) consistently were negative whereas tumor neovascularization commonly was VEGFR-3 positive. CONCLUSIONS: The results of the current study show that although VEGFR-3 shows specificity toward lymphatics in normal tissues, this receptor is distributed extensively in benign and malignant vascular tumors and therefore can be considered a novel marker in the assessment of endothelial cell differentiation of vascular neoplasms.

Papillary intralymphatic angioendothelioma (PILA): a report of twelve cases of a distinctive vascular tumor with phenotypic features of lymphatic vessels.

Six childhood vascular tumors were designated as "malignant endovascular papillary angioendothelioma" by Dabska in 1969. Since then, a few reports of similar cases were published, often called "Dabska tumors." Twelve similar cases were identified in review of vascular tumors from the authors' institutions. There were five men and seven women, including seven adults. Patient ages ranged from 8 to 59 years (mean, 30 years). The tumors occurred in the dermis or subcutis of the buttocks or thigh (n = 6), thumb or hand (n = 3), abdomen (n = 2), and heel (n = 1). The tumor sizes ranged from 1 to more than 40 cm (mean, 7.0 cm). The unifying feature of all cases was distinctive intravascular growth of well-differentiated endothelial cells presenting as a matchstick columnar configuration, sometimes with a large production of matrix that was positive for collagen type IV. In half the cases, these intravascular proliferations had an associated actin-positive pericytic proliferation. There was minimal cytologic atypia and rare to absent mitotic activity. Two cases had an adjacent lymphangioma, and two additional cases had clusters of lymphatic vessels adjacent to the tumor. All but two of the cases showed varying degrees of stromal or intraluminal lymphocytes. Occasional epithelioid endothelial cells were seen, but no cases had features typical of epithelioid, spindle cell, or retiform hemangioendothelioma. Tumor cells were positive for vimentin, von Willebrand factor, CD31, and focally for CD34 and were negative for keratins, epithelial membrane antigen, S-100 protein, and desmin. Vascular endothelial cell growth factor receptor type 3, a recently introduced marker for lymphatic endothelia, was positive in all eight cases that were studied, supporting a lymphatic phenotype. Follow-up in 8 of the 12 cases showed no evidence of recurrences, metastases, or residual disease during follow-ups ranging from 1 to 17 years (mean, 9 years). Based on the proliferative borderline features and the lymphatic phenotype, we propose to designate these tumors as papillary intralymphatic angioendothelioma. Additional cases with extensive follow-up should be studied to rule out variants with malignant potential.

Hobnail hemangioma ("targetoid hemosiderotic hemangioma"): clinicopathologic and immunohistochemical analysis of 62 cases.

Hobnail hemangioma, also known as "targetoid hemosiderotic hemangioma", represents a distinctive, benign vascular tumor, characterized histologically by a biphasic growth pattern of dilated vascular structures in the superficial dermis lined by prominent hobnail endothelial cells, and collagen dissecting, rather narrow neoplastic vessels in deeper parts of the lesion. We analyzed the clinicopathologic and immunohistochemical features in a series of 62 cases. Patient age range was 6-72 years (median: 32 years); 34 patients were male and 25 female. Clinically, a broad variation of diagnoses ranging from hemangioma to dermal melanocytic nevus and fibrous histiocytoma was suggested. Nineteen tumors arose in the lower and 13 in the upper extremities, 12 on the back, 8 in the buttock and hip region, and one case on the chest wall. Follow-up information on 35 patients (range from 1 to 4 years; mean: 1.5 years) revealed no local recurrence nor systemic metastasis. All neoplasms were located in the dermis and showed a broad morphologic spectrum in dependence of the age of the lesions. In addition to lesions resembling cavernous lymphangioma or lymphangioma circumscriptum, neoplasms were seen with morphologic features reminiscent to retiform hemangioendothelioma, progressive lymphangioma and so-called Dabska's tumor. Immunohistochemistry performed in 28 cases showed positive staining of tumor cells for CD31 in all cases tested, whereas only 3 out of 28 cases stained completely positive for CD34. In addition 4 out of 8 cases stained positively for vascular endothelial growth factor receptor-3 (VEGFR-3). Neoplastic endothelial cells were surrounded by actin-positive pericytes in only 7 out of 27 cases tested. Hobnail hemangioma occurs more frequently in male patients and arises commonly in the extremities and the trunk. Histologic and immunohistochemcial features suggest a lymphatic line of differentiation for this distinctive vascular neoplasm.

Endothelial growth factor receptors in human fetal heart.

BACKGROUND: Endothelial receptor tyrosine kinases include 3 members of the vascular endothelial growth factor receptor (VEGFR) family and 2 members of the angiopoietin receptor (Tie) family. In addition, the VEGF(165) isoform binds to neuropilin-1 (NP-1), a receptor for collapsins/semaphorins. The importance of these receptors for vasculogenesis and angiogenesis has been shown in gene-targeted mice, but so far, little is known about their exact expression patterns in the human vasculature. METHODS AND RESULTS: Frozen sections of human fetal heart were stained immunohistochemically with receptor-specific monoclonal (VEGFR, Tie) or polyclonal (NP-1) antibodies. The following patterns were observed: The endocardium was positive for VEGFR-1, VEGFR-2, NP-1, Tie-1, and Tie-2 but negative for VEGFR-3. The coronary vessels were positive for Tie-1, Tie-2, VEGFR-1, and NP-1 and negative for VEGFR-2 and VEGFR-3. Myocardial capillaries and epicardial blood vessels stained for VEGFR-1, VEGFR-2, NP-1, and Tie-1; myocardial capillaries and epicardial veins weakly for Tie-2; and epicardial lymphatic vessels for VEGFR-2 and VEGFR-3, weakly for Tie-1 and Tie-2, but not for VEGFR-1 or NP-1. CONCLUSIONS: The results demonstrate differential expression of the endothelial growth factor receptors in distinct types of vessels in the human heart. This information is useful for the understanding of their roles in physiological and pathological processes and for their diagnostic and therapeutic application in cardiovascular medicine.

Polarized vascular endothelial growth factor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaris. Evidence for a trophic paracrine relation.

The retinal pigment epithelium (RPE) maintains the choriocapillaris (CC) in the normal eye and is involved in the pathogenesis of choroidal neovascularization in age-related macular degeneration. Vascular endothelial growth factor-A (VEGF) is produced by differentiated human RPE cells in vitro and in vivo and may be involved in paracrine signaling between the RPE and the CC. We investigated whether there is a polarized secretion of VEGF by RPE cells in vitro. Also, the localization of VEGF receptors in the human retina was investigated. We observed that highly differentiated human RPE cells, cultured on transwell filters in normoxic conditions, produced two- to sevenfold more VEGF toward their basolateral side as compared to the apical side. In hypoxic conditions, VEGF-A secretion increased to the basal side only, resulting in a three- to 10-fold higher basolateral secretion. By immunohistochemistry in 30 human eyes and in two cynomolgus monkey eyes, KDR (VEGFR-2) and flt-4 (VEGFR-3) were preferentially localized at the side of the CC endothelium facing the RPE cell layer, whereas flt-1 (VEGFR-1) was found on the inner CC and on other choroidal vessels. Our results indicate that RPE secretes VEGF toward its basal side where its receptor KDR is located on the adjacent CC endothelium, suggesting a role of VEGF in a paracrine relation, possibly in cooperation with flt-4 and its ligand. This can explain the known trophic function of the RPE in the maintenance of the CC and its fenestrated permeable phenotype and points to a role for VEGF in normal eye functioning. Up-regulated basolateral VEGF secretion by RPE in hypoxia or loss of polarity of VEGF production may play a role in the pathogenesis of choroidal neovascularization.

Expression of vascular endothelial growth factor receptor-3 and podoplanin suggests a lymphatic endothelial cell origin of Kaposi's sarcoma tumor cells.

Despite intensive research over the past decade, the exact lineage relationship of Kaposi's sarcoma (KS) tumor cells has not yet been settled. In the present study, we investigated the expression of two markers for lymphatic endothelial cells (EC), ie, vascular endothelial growth factor receptor-3 (VEGFR-3) and podoplanin, in AIDS and classic KS. Both markers were strongly expressed by cells lining irregular vascular spaces in early KS lesions and by tumor cells in advanced KS. Double-staining experiments by confocal laser microscopy established that VEGFR-3-positive and podoplanin-positive cell populations were identical and uniformly expressed CD31. By contrast, these cells were negative for CD45, CD68, and PAL-E, excluding their hemopoietic and blood vessel endothelial cell nature. Podoplanin expression in primary KS tumor lysates was confirmed by Western blot analysis. Both splice variants of VEGFR-3 were found in KS-tumor-derived RNA by RT-PCR. In contrast to KS tumor cells in situ, no expression of VEGFR-3 and podoplanin was detected in any of four KS-derived spindle cell cultures and in one KS-derived autonomously growing cell line (KS Y-1). Our findings that KS tumor cells express two lymphatic EC markers in situ strongly suggest that they are related to or even derived from the lymphatic EC lineage. Lack of these antigens on cultured cells derived from KS lesions indicates that they might not represent tumor cells that grow in tissue culture, but rather other cell types present in KS lesions.

Expression of the vascular endothelial growth factor C receptor VEGFR-3 in lymphatic endothelium of the skin and in vascular tumors.

It is difficult to identify lymph vessels in tissue sections by histochemical staining, and thus a specific marker for lymphatic endothelial cells would be more practical in histopathological diagnostics. Here we have applied a specific antigenic marker for lymphatic endothelial cells in the human skin, the vascular endothelial growth factor receptor-3 (VEGFR-3), and show that it identifies a distinct vessel population both in fetal and adult skin, which has properties of lymphatic vessels. The expression of VEGFR-3 was studied in normal human skin by in situ hybridization, iodinated ligand binding, and immunohistochemistry. A subset of developing vessels expressed the VEGFR-3 mRNA in fetal skin as shown by in situ hybridization and radioiodinated vascular endothelial growth factor (VEGF)-C bound selectively to a subset of vessels in adult skin that had morphological characteristics of lymphatic vessels. Monoclonal antibodies against the extracellular domain of VEGFR-3 stained specifically endothelial cells of dermal lymph vessels, in contrast to PAL-E antibodies, which stained only blood vessel endothelia. In addition, staining for VEGFR-3 was strongly positive in the endothelium of cutaneous lymphangiomatosis, but staining of endothelial cells in cutaneous hemangiomas was weaker. These results establish the utility of anti-VEGFR-3 antibodies in the identification of lymphovascular channels in the skin and in the differential diagnosis of skin lesions involving lymphatic or blood vascular endothelium.

Lymphatic endothelium and Kaposi's sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor receptor-3.

Lymphatic vessels have been difficult to study in detail in normal and tumor tissues because of the lack of molecular markers. Here, monoclonal antibodies against the extracellular domain of the vascular endothelial growth factor-C receptor that we have named VEGFR-3 were found to specifically stain endothelial cells of lymphatic vessels and vessels around tumors such as lymphoma and in situ breast carcinoma. Interestingly, the spindle cells of several cutaneous nodular AIDS-associated Kaposi's sarcomas and the endothelium around the nodules were also VEGFR-3 positive. The first specific molecular marker for the lymphatic endothelium should provide a useful tool for the analysis of lymphatic vessels in malignant tumors and their metastases and the cellular origin and differentiation of Kaposi's sarcomas.