Johanna Arola

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 α 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.—Partanen, T. A., Arola, J., Saaristo, A., Jussila, L., Ora, A., Miettinen, M., Stacker, S. A., Achen, M. G., Alitalo, K. VEGF‐C and VEGF‐D expression in neuroendocrine cells and their receptor, VEGFR‐3, in fenestrated blood vessels in human tissues. FASEB J. 14, 2087–2096 (2000)

Familial Cutaneous Leiomyomatosis Is a Two-Hit Condition Associated with Renal Cell Cancer of Characteristic Histopathology

Little has been known about the molecular background of familial multiple cutaneous leiomyomatosis (MCL). We report here a clinical, histopathological, and molecular study of a multiple cutaneous leiomyomatosis kindred with seven affected members. This detailed study revealed strong features of a recently described cancer predisposition syndrome, hereditary leiomyomatosis and renal cell cancer (HLRCC). The family was compatible with linkage to the HLRCC locus in 1q. Also, all seven cutaneous leiomyomas derived from the proband and analyzed for loss of heterozygosity displayed loss of the wild-type allele, confirming the association with a susceptibility gene in chromosome 1q. One individual had had renal cell cancer at the age of 35 years. This tumor displayed a rare papillary histopathology, which appears to be characteristic for HLRCC. The derived linkage, loss of heterozygosity, and clinical data suggest that MCL and HLRCC are a single disease with a variable phenotype. The possibility that members of leiomyomatosis families are predisposed to renal cell cancer should be taken into account.

Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are important regulators of blood and lymphatic vessel growth and vascular permeability. The VEGF‐C/VEGFR‐3 signaling pathway is crucial for lymphangiogenesis, and heterozygous inactivating missense mutations of the VEGFR‐3 gene are associated with hereditary lymphedema. However, VEGF‐C can have potent effects on blood vessels because its receptor VEGFR‐3 is expressed in certain blood vessels and because the fully processed form of VEGF‐C also binds to the VEGFR‐2 of blood vessels. To characterize the in vivo effects of VEGF‐C on blood and lymphatic vessels, we have overexpressed VEGF‐C via adenovirus‐and adeno‐associated virus‐mediated transfection in the skin and respiratory tract of athymic nude mice. This resulted in dose‐dependent enlargement and tortuosity of veins, which, along with the collecting lymphatic vessels were found to express VEGFR‐2. Expression of angiopoietin 1 blocked the increased leakiness of the blood vessels induced by VEGF‐C whereas vessel enlargement and lymphangiogenesis were not affected. However, angiogenic sprouting of new blood vessels was not observed in response to AdVEGF‐C or AAV‐VEGF‐C. These results show that virally produced VEGF‐C induces blood vessel changes, including vascular leak, but its angiogenic potency is much reduced compared with VEGF in normal skin.—Saaristo, A., Veikkola, T., Enholm, B. Hytönen, M., Arola, J., Pajusola, K., Turunen, P., Jeltsch, M., Karkkainen, M. J., Kerjaschki, D., Bueler, H., Ylä‐Herttuala, S., Alitalo, K. Adenoviral VEGF‐C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes. FASEB J. 16, 1041–1049 (2002)

Hepatic Stearoyl-CoA Desaturase (SCD)-1 Activity and Diacylglycerol but Not Ceramide Concentrations Are Increased in the Nonalcoholic Human Fatty Liver

OBJECTIVE—To determine whether 1) hepatic ceramide and diacylglycerol concentrations, 2) SCD1 activity, and 3) hepatic lipogenic index are increased in the human nonalcoholic fatty liver. RESEARCH DESIGN AND METHODS—We studied 16 subjects with (n = 8) and without (n = 8) histologically determined nonalcoholic fatty liver (NAFL+ and NAFL−) matched for age, sex, and BMI. Hepatic concentrations of lipids and fatty acids were quantitated using ultra-performance liquid chromatography coupled to mass spectrometry and gas chromatography. RESULTS—The absolute (nmol/mg) hepatic concentrations of diacylglycerols but not ceramides were increased in the NAFL+ group compared with the NAFL− group. The livers of the NAFL+ group contained proportionally less long-chain polyunsaturated fatty acids as compared with the NAFL− group. Liver fat percent was positively related to hepatic stearoyl-CoA desaturase 1 (SCD1) activity index (r = 0.70, P = 0.003) and the hepatic lipogenic index (r = 0.54, P = 0.030). Hepatic SCD1 activity index was positively related to the concentrations of diacylglycerols (r = 0.71, P = 0.002) but not ceramides (r = 0.07, NS). CONCLUSIONS—We conclude that diacylglycerols but not ceramides are increased in NAFL. The human fatty liver is also characterized by depletion of long polyunsaturated fatty acids in the liver and increases in hepatic SCD1 and lipogenic activities.

Biallelic Inactivation of Fumarate Hydratase (FH) Occurs in Nonsyndromic Uterine Leiomyomas but Is Rare in Other Tumors

Germline mutations in the fumarate hydratase (FH) gene at 1q43 predispose to dominantly inherited cutaneous and uterine leiomyomas, uterine leiomyosarcoma, and papillary renal cell cancer (HLRCC syndrome). To evaluate the role of FH inactivation in sporadic tumorigenesis, we analyzed a series of 299 malignant tumors representing 10 different malignant tumor types for FH mutations. Additionally, 153 uterine leiomyomas from 46 unselected individuals were subjected to and informative in loss of heterozygosity analysis at the FH locus, and the five (3.3%) tumors displaying loss of heterozygosity were subjected to FH mutation analysis. Although mutation search in the 299 malignant tumors was negative, somatic FH mutations were found in two nonsyndromic leiomyomas; a splice site change IVS4 + 3A>G, leading to deletion of exon four, and a missense mutation Ala196Thr. The occurrence of somatic mutations strongly suggests that FH is a true target of the 1q43 deletions. Although uterine leiomyomas are the most common tumors of women, specific inactivating somatic mutations contributing to the formation of nonsyndromic leiomyomas have not been reported previously. Taking into account the apparent risk of uterine leiomyosarcoma associated with FH germline mutations, the finding raises the possibility that also some nonsyndromic leiomyomas may have a genetic profile that is more prone to malignant degeneration. Our data also indicate that somatic FH mutations appear to be limited to tumor types observed in hereditary leiomyomatosis and renal cell cancer.

LKB1 Somatic Mutations in Sporadic Tumors

Germline mutations of LKB1/Peutz-Jeghers syndrome gene predispose carriers to hamartomatous polyposis of the gastrointestinal tract as well as to cancer of different organ systems. Although Peutz-Jeghers syndrome patients frequently present with neoplasms of the colon, stomach, small intestine, pancreas, breast, ovaries, and cervix, somatic mutations appear to be rare in the sporadic tumor types thus far studied (colorectal, gastric, testicular, and breast cancers). To evaluate whether somatic mutations of LKB1 contribute to the tumorigenesis of yet unstudied tumor types, we screened 14 cell lines and 129 tumor specimens from different cancers for a genetic defect in LKB1. Six melanoma and eight myeloma cell lines were scrutinized for LKB1 somatic mutations by genomic sequencing. No changes were found in the coding LKB1 sequence and exon/intron boundaries. Next, we analyzed 12 pancreatic, 8 gastric, 12 ovarian granulosa cell, 26 cervical, 28 lung, 24 soft tissue, and 19 renal tumors by single-strand conformational polymorphism analysis. Three changes in LKB1 coding nucleotide sequence were identified. One base pair deletion at A957 and G958 substitution by T occurred in a cervical adenocarcinoma sample, resulting in a frameshift and premature stop codon at position 335. Substitution of A581 by T occurred in a lung adenocarcinoma sample, resulting in the change of aspartic acid at position 194 to valine. A loss of another allele was detected in this sample. One silent change, C1257T, was found in a pancreatic carcinoma sample. The changes were not present in the matched normal tissue DNA samples. Our results suggest that mutational inactivation of LKB1 is a rare event in most sporadic tumor types.

Comparison of Lipid and Fatty Acid Composition of the Liver, Subcutaneous and Intra-abdominal Adipose Tissue, and Serum

Ceramides may mediate saturated fat–induced insulin resistance, but there are no data comparing ceramide concentrations between human tissues. We therefore performed lipidomic analysis of human subcutaneous (SCfat) and intra‐abdominal (IAfat) adipose tissue, the liver, and serum in eight subjects. The liver contained (nmol/mg tissue) significantly more ceramides (1.5–3‐fold), sphingomyelins (7–8‐fold), phosphatidylethanolamines (10–11‐fold), lysophosphatidylcholines (7–12‐fold), less ether‐linked phosphatidylcholines (2–2.5‐fold) but similar amounts of diacylglycerols as compared to SCfat and IAfat. The amounts of ceramides and their synthetic precursors, such as palmitic (16:0) free fatty acids and sphingomyelins, differed considerably between the tissues. The liver contained proportionally more palmitic, stearic (18:0), and long polyunsaturated fatty acids than adipose tissues. Stearoyl‐CoA desaturase 1 (SCD1) activity reflected by serum, estimated from the 16:1/16:0‐ratio, was closely related to that in the liver (r = 0.86, P = 0.024) but not adipose tissues. This was also true for estimated elongase (18:1/16:1, r = 0.89, P = 0.01), and Δ5 (20:4/20:3, r = 0.89, P = 0.012) and Δ6 (18:3[n‐6]/18:2, r = 1.0, P < 0.001) desaturase activities. We conclude that the human liver contains higher concentrations of ceramides and saturated free fatty acids than either SCfat or IAfat.

High Mobility Group Box 1 Protein as a Marker of Hepatocellular Injury in Human Liver Transplantation

High mobility group box 1 protein (HMGB1), a cytokine actively secreted by phagocytes and passively released from necrotic cells, is an inflammatory mediator in experimental hepatic ischemia/reperfusion injury. We characterized its expression in human liver transplantation. In 20 patients, in addition to systemic samples, blood was drawn from portal and hepatic veins during and after reperfusion to assess changes within the graft. Plasma HMGB1, tumor necrosis factor α (TNF‐α), and interleukin‐6 (IL‐6) levels were measured, and HMGB1 immunohistochemistry was performed on biopsies taken before and after reperfusion. Plasma HMGB1 was undetectable before reperfusion, and levels in systemic circulation peaked after graft reperfusion. At portal declamping, HMGB1 levels were substantially higher in the caval effluent [188 (80‐371) ng/mL] than in portal venous blood [0 (0‐3) ng/mL, P < 0.001]. HMGB1 release from the graft continued thereafter. HMGB1 levels were not related to TNF‐α or IL‐6 levels. HMGB1 expression was up‐regulated in biopsies taken after reperfusion (P = 0.020), with intense hepatocyte and weak neutrophil staining. HMGB1 levels in hepatic venous blood correlated with graft steatosis (r = 0.497, P = 0.03) and peak postoperative alanine aminotransferase levels (r = 0.588, P = 0.008). Our results indicate that HMGB1 originates from the graft and is a marker of hepatocellular injury in human liver transplantation. Liver Transpl 14:1517–1525, 2008.

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.

The expression of AIP-related molecules in elucidation of cellular pathways in pituitary adenomas.

Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene predispose to the development of pituitary adenomas. Here, we characterized AIP mutation positive (AIPmut+) and AIP mutation negative (AIPmut-) pituitary adenomas by immunohistochemistry. The expressions of the AIP-related proteins aryl hydrocarbon receptor (AHR), AHR nuclear translocator (ARNT), cyclin-dependent kinase inhibitor 1B encoding p27(Kip1), and hypoxia-inducible factor 1-alpha were examined in 14 AIPmut+ and 53 AIPmut- pituitary adenomas to detect possible expression differences. In addition, the expression of CD34, an endothelial and hematopoietic stem cell marker, was analyzed. We found ARNT to be less frequently expressed in AIPmut+ pituitary adenomas (P = 0.001), suggesting that AIP regulates the ARNT levels. AIP small interfering RNA-treated HeLa, HEK293, or Aip-null mouse embryonic fibroblast cells did not show lowered expression of ARNT. Instead, in the pituitary adenoma cell line GH3, Aip silencing caused a partial reduction of Arnt and a clear increase in cell proliferation. We also observed a trend for increased expression of nuclear AHR in AIPmut+ samples, although the difference was not statistically significant (P = 0.06). The expressions of p27(Kip1), hypoxia-inducible factor 1-alpha, or CD34 did not differ between tumor types. The present study shows that the expression of ARNT protein is significantly reduced in AIPmut+ tumors. We suggest that the down-regulation of ARNT may be connected to an imbalance in AHR/ARNT complex formation arising from aberrant cAMP signaling.