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Renal Angiomyolipomas from Patients with Sporadic Lymphangiomyomatosis Contain Both Neoplastic and Non-Neoplastic Vascular Structures

2003; Elsevier BV; Volume: 162; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)63843-6

1525-2191

Magdalena Karbowniczek, Jane Yu, Elizabeth P. Henske,

Renal and related cancers

Renal angiomyolipomas are highly vascular tumors that occur sporadically, in women with pulmonary lymphangiomyomatosis (LAM), and in tuberous sclerosis complex (TSC). The goal of this study was to determine whether the distinctive vessels of angiomyolipomas are neoplastic or reactive. We studied angiomyolipomas with loss of heterozygosity (LOH) in the TSC2 region of chromosome 16p13 from patients with LAM. We found that angiomyolipomas contain five morphologically distinct vessel types: cellular, collagenous, hemangiopericytic, glomeruloid, and aneurysmatic. Using laser capture microdissection, we determined that four of the vessel types have TSC2 LOH and are therefore neoplastic. One vessel type, collagenous vessels, did not have LOH, and is presumably reactive. Recently, activation of S6 Kinase and its target S6 ribosomal protein (S6) was demonstrated in cells lacking TSC2 expression. We found that angiomyolipoma vessel types in which LOH were detected were immunoreactive with anti-phospho-S6 antibodies. Angiomyolipoma cells without LOH, including the endothelial component of the vessels, were not immunoreactive. To our knowledge, angiomyolipomas are the first benign vascular tumor in which the vascular cells, rather than the stromal cells, have been found to be neoplastic. Angiomyolipomas appear to reflect novel vascular mechanisms that may be the result of activation of cellular pathways involving S6 Kinase. Renal angiomyolipomas are highly vascular tumors that occur sporadically, in women with pulmonary lymphangiomyomatosis (LAM), and in tuberous sclerosis complex (TSC). The goal of this study was to determine whether the distinctive vessels of angiomyolipomas are neoplastic or reactive. We studied angiomyolipomas with loss of heterozygosity (LOH) in the TSC2 region of chromosome 16p13 from patients with LAM. We found that angiomyolipomas contain five morphologically distinct vessel types: cellular, collagenous, hemangiopericytic, glomeruloid, and aneurysmatic. Using laser capture microdissection, we determined that four of the vessel types have TSC2 LOH and are therefore neoplastic. One vessel type, collagenous vessels, did not have LOH, and is presumably reactive. Recently, activation of S6 Kinase and its target S6 ribosomal protein (S6) was demonstrated in cells lacking TSC2 expression. We found that angiomyolipoma vessel types in which LOH were detected were immunoreactive with anti-phospho-S6 antibodies. Angiomyolipoma cells without LOH, including the endothelial component of the vessels, were not immunoreactive. To our knowledge, angiomyolipomas are the first benign vascular tumor in which the vascular cells, rather than the stromal cells, have been found to be neoplastic. Angiomyolipomas appear to reflect novel vascular mechanisms that may be the result of activation of cellular pathways involving S6 Kinase. Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome characterized by seizures, mental retardation, autism, and tumors in the brain, retina, kidney, heart, and skin. Angiomyolipomas are benign tumors with three distinct components: smooth muscle cells, adipose cells, and abnormal blood vessels.1Eble JN Angiomyolipoma of kidney.Semin Diagn Pathol. 1998; 15: 21-40PubMed Google Scholar Mutations in two genes, TSC1 on chromosome 9q342van Slegtenhorst M de Hoogt R Hermans C Nellist M Janssen B Verhoef S Lindhout D van den Ouweland A Halley D Young J Burley M Jeremiah S Woodward K Nahmias J Fox M Ekong R Osborne J Wolfe J Povey S Snell R Cheadle J Jones A Tachataki M Ravine D Sampson J Reeve M Richardson P Wilmer R Munro C Hawkins T Sepp T Ali J Ward S Green A Yates J Kwiatkowska J Henske E Short M Haines J Jozwiak S Kwiatkowski D Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34.Science. 1997; 277: 805-808Crossref PubMed Scopus (1430) Google Scholar and TSC2 chromosome 16p13,3European Chromosome 16 Tuberous Sclerosis Consortium Identification and characterization of the tuberous sclerosis gene on chromosome 16.Cell. 1993; 73: 1305-1315Google Scholar cause TSC. Loss of heterozygosity (LOH) in the TSC1 or TSC2 region occurs in most angiomyolipomas, rhabdomyomas, and astrocytomas from TSC patients.4Henske EP Scheithauer BW Short MP Wollmann R Nahmias J Hornigold N van Slegtenhorst M Welsh CT Kwiatkowski DJ Allelic loss is frequent in tuberous sclerosis kidney lesions but rare in brain lesions.Am J Hum Genet. 1996; 59: 400-406PubMed Google Scholar TSC2 LOH also occurs in 10% of sporadic angiomyolipomas5Henske EP Neumann HP Scheithauer BW Herbst EW Short MP Kwiatkowski DJ Loss of heterozygosity in the tuberous sclerosis (TSC2) region of chromosome band 16p13 occurs in sporadic as well as TSC-associated renal angiomyolipomas.Genes Chromosomes Cancer. 1995; 13: 295-298Crossref PubMed Scopus (229) Google Scholar and in 60% of angiomyolipomas from women with the sporadic form of lymphangiomyomatosis (LAM).6Smolarek TA Wessner LL McCormack FX Mylet JC Menon AG Henske EP Evidence that lymphangiomyomatosis is caused by TSC2 mutations: chromosome 16p13 loss of heterozygosity in angiomyolipomas and lymph nodes from women with lymphangiomyomatosis.Am J Hum Genet. 1998; 62: 810-815Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar Although it is known that the smooth muscle and fat components of angiomyolipomas have TSC1 or TSC2 LOH, whether the dysplastic vessels within the angiomyolipomas have LOH is an area of uncertainty. We have previously found that angiomyolipoma vessels from two TSC patients did not contain the second hit somatic genetic event, indicating that they are not neoplastic.7Henske EP Wessner LL Golden J Scheithauer BW Vortmeyer AO Zhuang Z Klein-Szanto AJ Kwiatkowski DJ Yeung RS Loss of tuberin in both subependymal giant cell astrocytomas and angiomyolipomas supports a two-hit model for the pathogenesis of tuberous sclerosis tumors.Am J Pathol. 1997; 151: 1639-1647PubMed Google Scholar Recently, however, another group found the opposite result in one angiomyolipoma from a TSC patient.8Niida Y Stemmer-Rachamimov AO Logrip M Tapon D Perez R Kwiatkowski DJ Sims K MacCollin M Louis DN Ramesh V Survey of somatic mutations in tuberous sclerosis complex (tsc) hamartomas suggests different genetic mechanisms for pathogenesis of tsc lesions.Am J Hum Genet. 2001; 69: 493-503Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar Here, we studied angiomyolipomas from patients with the sporadic form of LAM. In these angiomyolipomas, we identified five distinct morphological types of vessels. Four of the vessel types had TSC2 LOH, and are neoplastic. One vessel type lacked TSC2 LOH, and is therefore non-neoplastic. The endothelial cells also lacked LOH. All of the neoplastic components of the tumor, as defined by the LOH analysis, showed hyperphosphorylation of the ribosomal protein S6 relative to the non-neoplastic components of the tumor. Angiomyolipomas may be the first example of a human tumor in which formation of blood vessel walls by tumor cells has been demonstrated. This study was approved by the Institutional Review Board of Fox Chase Cancer Center. All four patients (patients 436, 437, 487, and 492) have the sporadic form of lymphangiomyomatosis and each had a single renal angiomyolipoma. The patients ranged in age from 20 to 39 years at the time of angiomyolipoma resection. The angiomyolipomas had maximum dimensions ranging from 9.5 to 22 cm. Loss of heterozygosity in these angiomyolipomas has been previously reported.6Smolarek TA Wessner LL McCormack FX Mylet JC Menon AG Henske EP Evidence that lymphangiomyomatosis is caused by TSC2 mutations: chromosome 16p13 loss of heterozygosity in angiomyolipomas and lymph nodes from women with lymphangiomyomatosis.Am J Hum Genet. 1998; 62: 810-815Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar Paraffin sections were deparaffinized and rehydrated. For antigen retrieval, sections were boiled in Citric Buffer (10 mmol/L sodium citrate-trisodium salt dihydrate, Sigma, St. Louis, MO), pH 6.0, at 750 W for 10 minutes. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 minutes at room temperature. Non-specific background was eliminated by incubating the tissue with normal goat serum (Super Sensitive Kit, BioGenex, San Ramon, CA) for 30 minutes at room temperature. The sections were then incubated in a humidified chamber with mouse monoclonal antibodies against desmin, vimentin, muscle-specific actin (α and γ isotypes, all from BioGenex), or rabbit polyclonal antibodies against phospho-S6 ribosomal protein (Cell Signaling Technology, Beverly, MA), then rinsed and incubated with biotinylated goat anti-mouse antibody (BioGenex) for 30 minutes at room temperature. Visualization was performed using streptavidin-peroxidase (BioGenex). Sections were counterstained with Gill's hematoxylin. Slides were prepared with Masson trichrome staining for evaluation of collagen deposition and with periodic acid-Schiff (PAS) stain (with and without diastase) for evaluation of glycogen deposition, using standard methods. Different types of vessels were identified morphologically on hematoxylin and eosin (H&E) stained slides. Laser capture microdissection (PixCell II, Arcturus Engineering, Mountain View, CA) was used to isolate cells from the smooth muscle and fat components of the angiomyolipoma, and cells from the walls of each vessel type. The endothelial cell layer was avoided (Figure 1,A and B). For the angiomyolipoma from patient 437, we were able to separately capture endothelial cells from collagenous, cellular, and aneurysmatic vessels. For all specimens, DNA was extracted by overnight incubation in 30 μl of extraction buffer (0.5% Tween 20, 0.2 mg/ml Proteinase K, 0.05 mol/L Tris-HCl (pH 8.9), 2 mmol/L EDTA, and 1.0 mmol/L NaCl). A panel of microsatellite markers near the TSC2 locus on chromosome 16p13 was used: D16S287, D16S291, D16S418, D16S749, and Kg8 (Research Genetics, Huntsville, AL). LOH analyses were performed using a 2.5 μl aliquot of the DNA solution in a 10-μl polymerase chain reaction.4Henske EP Scheithauer BW Short MP Wollmann R Nahmias J Hornigold N van Slegtenhorst M Welsh CT Kwiatkowski DJ Allelic loss is frequent in tuberous sclerosis kidney lesions but rare in brain lesions.Am J Hum Genet. 1996; 59: 400-406PubMed Google Scholar The PCR amplification consisted of 95°C for 5 minutes followed by 40 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 45 seconds, and a final extension at 72°C for 10 minutes. PCR was performed with radioactive phosphorus-labeled deoxyguanosine triphosphate in the reaction mix. TaqStart antibody (Clontech, Palo Alto, CA) at final concentration of 0.056 μmol/L was used to enhance specificity. The PCR products were resolved by denaturating 8 mol/L urea polyacrylamide gel electrophoresis (Gibco, Grand Island, NY) and were visualized by autoradiography. All results were repeated at least twice for confirmation. In H&E stained sections, we identified five morphological types of vessels in the angiomyolipomas. Two of the vessel types, which we will refer to as cellular vessels and collagenous vessels, have thick walls and large lumens. Cellular vessels have thick, highly cellular walls (Figure 1A). Collagenous vessels have hypocellular walls with abundant extracellular material (Figure 1B). Three of the four angiomyolipomas in this study contained cellular vessels and three of the four contained collagenous vessels. The cellular and collagenous vessels were large, with a typical wall thickness of 200 to 400 μm and lumen size of 500 to 800 μm. The walls of cellular vessels usually contained cells with clear cytoplasm and a spindle or polygonal shape, similar in appearance to the angiomyolipoma's smooth muscle component. In contrast, cells in the walls of collagenous vessels tended to be more elongated, with dense pink cytoplasm. Collagenous angiomyolipoma vessels have been previously described,1Eble JN Angiomyolipoma of kidney.Semin Diagn Pathol. 1998; 15: 21-40PubMed Google Scholar, 9Murphy W Beckwith J Farrow G Tumors of the kidney, bladder, and related urinary structures.in: Rosai J Atlas of Tumor Pathology. Armed Forces Institute of Pathology, Washington DC1994: 92-118Google Scholar but we did not find previous reports of cellular vessels. In some collagenous vessels we observed the apparent infiltration into the vessel wall of larger cells with the appearance of the angiomyolipoma's smooth muscle cell component (Figure 2, A and B). These cells were present to varying degrees in about 30% of collagenous vessels. The relationship of these larger cells to the tendency of benign angiomyolipomas to spread to regional lymph nodes1Eble JN Angiomyolipoma of kidney.Semin Diagn Pathol. 1998; 15: 21-40PubMed Google Scholar and the possible spread of angiomyolipoma cells to the lung in patients with sporadic LAM10Carsillo T Astrinidis A Henske EP Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis.Proc Natl Acad Sci USA. 2000; 97: 6085-6090Crossref PubMed Scopus (560) Google Scholar is not known. Three types of vessels with thinner walls were identified. The first, hemangiopericytic vessels (Figure 1C), were often branching, with typical wall thickness around 50 μm. These vessels were identified in three of the angiomyolipomas. Glomeruloid vessels (Figure 1D) are often present within the fat component of angiomyolipoma. Glomeruloid vessels have the smallest lumen of any of the vessel types (typically 50 to 80 μm) with a cellular wall with thickness of 40 to 50 μm. The third type of thin-walled vessel, aneurysmatic vessels (Figure 1E) have fibrous, hypocellular walls, which sometimes create small dilatations or aneurysms, with typical wall thickness between 60 to 80 μm. Hemangiopericytic and glomeruloid vessels have been previously described only in atypical epithelioid angiomyolipomas from patients without TSC.11Delgado R de Leon Bojorge B Albores-Saavedra J A typical angiomyolipoma of the kidney: a distinct morphologic variant that is easily confused with a variety of malignant neoplasms.Cancer. 1998; 83: 1581-1592Crossref PubMed Scopus (67) Google Scholar Aneurysmatic and glomeruloid vessels were each identified in two of the angiomyolipomas. Masson trichrome staining revealed abundant collagen deposition in the wall of the collagenous (Figure 2C) and aneurysmatic vessels (data not shown), but not cellular vessels (Figure 2D), glomeruloid vessels, or hemangiopericytic vessels. PAS staining revealed fine, diastase-negative glycogen granules deposited in the angiomyolipoma's smooth muscle component and in the cells lining all five types of vessels (data not shown). The angiomyolipoma's smooth muscle cell and fat components and the cells within all five vessel types were immunoreactive with muscle-specific actin and with vimentin (data not shown). Desmin immunoreactivity was present in the smooth muscle and adipose components of the angiomyolipomas, as well as in the cells lining the cellular vessels (Figure 2F) and the hemangiopericytic vessels, but not the collagenous (Figure 2E), aneurysmatic, or glomeruloid vessels. Smooth muscle cells from normal renal blood vessels were immunoreactive for vimentin, desmin, and muscle-specific actin. The immunohistochemical features of the different vessel types are summarized in Table 1 and illustrated in Figure 3.Table 1Histochemical and Immunohistochemical Comparison of Angiomyolipoma Cell and Vessel TypesCell type or vessel typeCollagen depositionGlycogen depositionVimentinMuscle-specific actinDesminHMB-45Phospho-S6 ribosomal proteinAngiomyolipoma smooth muscle cells+ focally between stromal cells++++++Adipose tissue−+ focally+++++Cellular vessels−++++++Hemangiopericytic vessels−++++++Glomeruloid vessels−+++−++Aneurysmatic vessels++++−+ focally+Collagenous vessels++++−−−Normal renal blood vessels−−+++−− Open table in a new tab Each of the angiomyolipomas in this study was previously found to have LOH in the TSC2 region of chromosome 16p13.6Smolarek TA Wessner LL McCormack FX Mylet JC Menon AG Henske EP Evidence that lymphangiomyomatosis is caused by TSC2 mutations: chromosome 16p13 loss of heterozygosity in angiomyolipomas and lymph nodes from women with lymphangiomyomatosis.Am J Hum Genet. 1998; 62: 810-815Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar Laser capture microdissected specimens of smooth muscle and adipose components were analyzed separately for LOH using a panel of microsatellite markers near the TSC2 gene. In each case, the angiomyolipoma smooth muscle cell and adipose components had LOH for multiple markers. We next analyzed microdissected cells from the wall of each vessel type for LOH (Figure 4, Table 2). The angiomyolipoma from patient 436 contained cellular vessels, hemangiopericytic vessels, and collagenous vessels. The cellular and hemangiopericytic vessels had LOH at four markers, and the collagenous vessels did not have LOH. The angiomyolipoma from patient 437 contained cellular vessels, hemangiopericytic vessels, glomeruloid vessels, and collagenous vessels. The cellular, hemangiopericytic, and glomeruloid vessels had LOH at two markers, while the collagenous vessels did not have LOH. The angiomyolipoma from patient 487 contained cellular vessels and aneurysmatic vessels, both of which had LOH at three markers. The angiomyolipoma from patient 492 contained aneurysmatic vessels, hemangiopericytic vessels, glomeruloid vessels, and collagenous vessels. The aneurysmatic, hemangiopericytic, and glomeruloid vessels had LOH at two markers, and the collagenous vessels did not have LOH.Table 2Loss of Heterozygosity Results for Individual Chromosome 16p13 MarkersPatientTissueD16S749D16S287D16S418D16S291Kg8436Angiomyolipoma*Non-microdissected tissue;—†vessels of this type not present in this case, or this marker not typed for this patients;LOH‡LOH: loss of heterozygosity;LOHLOHLOHSmooth muscle cells—LOHLOHLOHLOHAdipose tissue—LOHLOHLOHLOHCellular vessels—LOHLOHLOHLOHHemangiopericytic vessels—LOHLOHLOHLOHGlomeruloid vessels—————Aneurysmatic vessels—————Collagenous vessels—2§2: retention of heterozygosity;222437Angiomyolipoma*Non-microdissected tissue;222LOHLOHSmooth muscle cells222LOHLOHAdipose tissue—————Cellular vessels222LOHLOHHemangiopericytic vessels222LOHLOHGlomeruloid vessels222LOHLOHAneurysmatic vessels—————Collagenous vessels22222Endothelial cells———22487Angiomyolipoma*Non-microdissected tissue;LOH1¶1: non-informative marker.LOH1LOHSmooth muscle cellsLOH1LOH1LOHAdipose tissue—————Cellular vesselsLOH1LOH1LOHHemangiopericytic vessels—————Glomeruloid vessels—————Aneurysmatic vesselsLOH1LOH1LOHCollagenous vessels—————492Angiomyolipoma*Non-microdissected tissue;—2LOHLOH1Smooth muscle cells—————Adipose tissue—2LOHLOH1Cellular vessels—————Hemangiopericytic vessels—2LOHLOH1Glomeruloid vessels—2LOHLOH1Aneurysmatic vessels—2LOHLOH1Collagenous vessels—2221The markers are listed in centromeric to telomeric order.* Non-microdissected tissue;† vessels of this type not present in this case, or this marker not typed for this patients;‡ LOH: loss of heterozygosity;§ 2: retention of heterozygosity;¶ 1: non-informative marker. Open table in a new tab The markers are listed in centromeric to telomeric order. We next analyzed microdissected endothelial cells from three vessel types. Because of the need to collect a sufficient number of cells (at least 250 nuclei) for reliable LOH analyses, we were limited to angiomyolipomas with enough vessels of a single type to allow pooling of endothelial cells from different vessels. Therefore, the endothelial cell analysis was performed only on cellular, aneurysmatic, and collagenous vessels from patient 437. LOH was not present in the endothelial cells (Figure 5). In summary, for each of the angiomyolipomas, LOH was present in the smooth muscle cells lining cellular, hemangiopericytic, aneurysmatic, and glomeruloid vessels (Table 2), indicating that they are part of the neoplastic component of the tumor. In contrast, endothelial cells from cellular and aneurysmatic vessels did not have LOH, suggesting that the endothelial component of these vessels arises independently. LOH was not present in collagenous vessels, or in the endothelial cells lining collagenous vessels. Recently, S6 hyperphosphorylation was found in cells lacking tuberin expression12Goncharova EA Goncharov DA Eszterhas A Hunter DS Glassberg MK Yeung RS Walker CL Noonan D Kwiatkowski DJ Chou MM Panettieri Jr, RA Krymskaya VP Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation: a role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis (LAM).J Biol Chem. 2002; 277: 30958-30967Crossref PubMed Scopus (378) Google Scholar and in cells lacking hamartin expression,13Kwiatkowski DJ Zhang H Bandura JL Heiberger KM Glogauer M el-Hashemite N Onda H A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in Tsc1 null cells.Hum Mol Genet. 2002; 11: 525-534Crossref PubMed Scopus (547) Google Scholar indicating that the tuberin-hamartin complex negatively regulates the activity of S6 Kinase. We immunostained the angiomyolipomas with an antibody specific for phospho-S6. The cell types in which LOH was detected (the angiomyolipoma fat, smooth muscle cells and the cells lining the cellular, hemangiopericytic, glomeruloid, and aneurysmatic vessels) (Figure 6, A and B, D–F) were immunoreactive with anti-phospho S6 antibody. The endothelial cells lining these vessels, which did not have LOH, were not immunoreactive. The collagenous vessels, which did not have LOH, were also not immunoreactive (Figure 6C). TSC is a tumor suppressor gene disorder in which two of the most frequent tumors, renal angiomyolipomas and facial angiofibromas, have a prominent vascular component. Whether these vessels are reactive (resulting from the proliferation of normal cells in response to tumor-secreted factors) or neoplastic (consisting of tumor cells) is controversial, with prior studies providing conflicting data.7Henske EP Wessner LL Golden J Scheithauer BW Vortmeyer AO Zhuang Z Klein-Szanto AJ Kwiatkowski DJ Yeung RS Loss of tuberin in both subependymal giant cell astrocytomas and angiomyolipomas supports a two-hit model for the pathogenesis of tuberous sclerosis tumors.Am J Pathol. 1997; 151: 1639-1647PubMed Google Scholar, 8Niida Y Stemmer-Rachamimov AO Logrip M Tapon D Perez R Kwiatkowski DJ Sims K MacCollin M Louis DN Ramesh V Survey of somatic mutations in tuberous sclerosis complex (tsc) hamartomas suggests different genetic mechanisms for pathogenesis of tsc lesions.Am J Hum Genet. 2001; 69: 493-503Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar We examined the genetic basis of angiomyolipoma vessels in patients with the sporadic form of pulmonary lymphangiomyomatosis (LAM). Sporadic LAM angiomyolipomas frequently have somatic inactivation of both alleles of the TSC2 gene.6Smolarek TA Wessner LL McCormack FX Mylet JC Menon AG Henske EP Evidence that lymphangiomyomatosis is caused by TSC2 mutations: chromosome 16p13 loss of heterozygosity in angiomyolipomas and lymph nodes from women with lymphangiomyomatosis.Am J Hum Genet. 1998; 62: 810-815Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 10Carsillo T Astrinidis A Henske EP Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis.Proc Natl Acad Sci USA. 2000; 97: 6085-6090Crossref PubMed Scopus (560) Google Scholar We were surprised to find that the angiomyolipomas contained five vessel types, one of which (cellular) has not to our knowledge been previously recognized and two of which (glomeruloid and hemangiopericytic) have been previously recognized only within sporadic atypical epithelioid angiomyolipomas.11Delgado R de Leon Bojorge B Albores-Saavedra J A typical angiomyolipoma of the kidney: a distinct morphologic variant that is easily confused with a variety of malignant neoplasms.Cancer. 1998; 83: 1581-1592Crossref PubMed Scopus (67) Google Scholar The angiomyolipoma vessels had clear morphological differences in diameter, wall thickness, lumen size, aneurysmic wall dilatations, collagen deposition, and glycogen deposition. These differences may indicate that the TSC proteins play a functional role in the normal development and maturation of blood vessels. We separately microdissected cells from the walls of each vessel type, as well as cells in the smooth muscle and fat components of the angiomyolipomas, and analyzed the DNA for LOH in the TSC2 region of chromosome 16p13. The smooth muscle and fat components of the angiomyolipomas had LOH, as expected.7Henske EP Wessner LL Golden J Scheithauer BW Vortmeyer AO Zhuang Z Klein-Szanto AJ Kwiatkowski DJ Yeung RS Loss of tuberin in both subependymal giant cell astrocytomas and angiomyolipomas supports a two-hit model for the pathogenesis of tuberous sclerosis tumors.Am J Pathol. 1997; 151: 1639-1647PubMed Google Scholar, 8Niida Y Stemmer-Rachamimov AO Logrip M Tapon D Perez R Kwiatkowski DJ Sims K MacCollin M Louis DN Ramesh V Survey of somatic mutations in tuberous sclerosis complex (tsc) hamartomas suggests different genetic mechanisms for pathogenesis of tsc lesions.Am J Hum Genet. 2001; 69: 493-503Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar Four of the five vessel types (cellular, hemangiopericytic, glomeruloid, and aneurysmatic) also had LOH, and in each case the LOH pattern was identical to the LOH pattern in the smooth muscle and fat cells. This indicates that the cells in the walls of these vessels are neoplastic, and supports a model in which angiomyolipomas are derived from a mesenchymal cell that retains the ability to differentiate into multiple different lineages.14Stone CH Lee MW Amin MB Yaziji H Gown AM Ro JY Tetu B Paraf F Zarbo RJ Renal angiomyolipoma: further immunophenotypic characterization of an expanding morphologic spectrum.Arch Pathol Lab Med. 2001; 125: 751-758PubMed Google Scholar In contrast, collagenous vessels did not have LOH, and LOH was not detected in endothelial cells from cellular, aneurysmatic vessels, or collagenous vessels. The lack of LOH in the endothelial cells may be related to the previously demonstrated expression of vascular endothelial growth factor (VEGF) by angiomyolipomas,15Arbiser JL Brat D Hunter S D'Armiento J Henske EP Arbiser ZK Bai X Goldberg G Cohen C Weiss SW Tuberous sclerosis-associated lesions of the kidney, brain, and skin are angiogenic neoplasms.J Am Acad Dermatol. 2002; 46: 376-380Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar which could be involved in the recruitment of endothelial cells. Taken together, these results demonstrate that multiple genetic mechanisms contribute to angiomyolipoma blood vessel formation, including both neoplastic and non-neoplastic vessel wall formation and the recruitment of non-neoplastic endothelial cells. To our knowledge, this is the first time that the vascular component of a benign vascular tumor has been shown to be neoplastic.16Marchuk DA Pathogenesis of hemangioma.J Clin Invest. 2001; 107: 665-666Crossref PubMed Scopus (72) Google Scholar Studies of von Hippel Lindau-associated hemangioblastomas and retinal angiomas have consistently demonstrated that the stromal cell component, and not the vascular component, contains the second hit mutation.17Vortmeyer AO Gnarra JR Emmert-Buck MR Katz D Linehan WM Oldfield EH Zhuang Z von Hippel-Lindau gene deletion detected in the stromal cell component of a cerebellar hemangioblastoma associated with von Hippel-Lindau disease.Hum Pathol. 1997; 28: 540-543Abstract Full Text PDF PubMed Scopus (171) Google Scholar, 18Lee JY Dong SM Park WS Yoo NJ Kim CS Jang JJ Chi JG Zbar B Lubensky IA Linehan WM Vortmeyer AO Zhuang Z Loss of heterozygosity and somatic mutations of the VHL tumor suppressor gene in sporadic cerebellar hemangioblastomas.Cancer Res. 1998; 58: 504-508PubMed Google Scholar, 19Chan CC Vortmeyer AO Chew EY Green WR Matteson DM Shen DF Linehan WM Lubensky IA Zhuang Z VHL gene deletion and enhanced VEGF gene expression detected in the stromal cells of retinal angioma.Arch Ophthalmol. 1999; 117: 625-630Crossref PubMed Scopus (124) Google Scholar TSC, therefore, appears to reflect a novel genetic mechanism of blood vessel formation. Tuberin, the TSC2 gene product,20Consortium TECTS Identification and characterization of the tuberous sclerosis gene on chromosome 16: The European Chromosome 16 Tuberous Sclerosis Consortium.Cell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1534) Google Scholar and hamartin, the TSC1 gene product,2van Slegtenhorst M de Hoogt R Hermans C Nellist M Janssen B Verhoef S Lindhout D van den Ouweland A Halley D Young J Burley M Jeremiah S Woodward K Nahmias J Fox M Ekong R Osborne J Wolfe J Povey S Snell R Cheadle J Jones A Tachataki M Ravine D Sampson J Reeve M Richardson P Wilmer R Munro C Hawkins T Sepp T Ali J Ward S Green A Yates J Kwiatkowska J Henske E Short M Haines J Jozwiak S Kwiatkowski D Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34.Science. 1997; 277: 805-808Crossref PubMed Scopus (1430) Google Scholar have been shown to interact21Plank TL Yeung RS Henske EP Hamartin, the product of the tuberous sclerosis 1 (TSC1) gene, interacts with tuberin and appears to be localized to cytoplasmic vesicles.Cancer Res. 1998; 58: 4766-4770PubMed Google Scholar, 22van Slegtenhorst M Nellist M Nagelkerken B Cheadle J Snell R van den Ouweland A Reuser A Sampson J Halley D van der Sluijs P Interaction between hamartin and tuberin, the TSC1 and TSC2 gene products.Hum Mol Genet. 1998; 7: 1053-1057Crossref PubMed Scopus (500) Google Scholar and appear to be involved in multiple cellular pathways. Hamartin interacts with the ezrin-radixin-moesin (ERM) family of cytoskeletal proteins and activates the GTPase Rho.23Lamb RF Roy C Diefenbach TJ Vinters HV Johnson MW Jay DG Hall A The TSC1 tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase Rho.Nat Cell Biol. 2000; 2: 281-287Crossref PubMed Scopus (279) Google Scholar Since Rho is known to regulate vascular smooth muscle cell contraction, differentiation, and proliferation,24Laufs U Marra D Node K Liao JK 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors attenuate vascular smooth muscle proliferation by preventing rho GTPase-induced down-regulation of p27(Kip1).J Biol Chem. 1999; 274: 21926-21931Crossref PubMed Scopus (386) Google Scholar, 25van Nieuw Amerongen GP van Hinsbergh VW Cytoskeletal effects of rho-like small guanine nucleotide-binding proteins in the vascular system.Arterioscler Thromb Vasc Biol. 2001; 21: 300-311Crossref PubMed Scopus (136) Google Scholar, 26Begum N Sandu OA Ito M Lohmann SM Smolenski A Active Rho kinase (ROK-α) associates with insulin receptor substrate-1 and inhibits insulin signaling in vascular smooth muscle cells.J Biol Chem. 2002; 277: 6214-6222Crossref PubMed Scopus (144) Google Scholar it is interesting to speculate that aberrant signaling involving Rho contributes to blood vessel formation in angiomyolipomas. Recently, hyperphosphorylation of p70 S6 Kinase (S6K) and its substrate ribosomal protein S6 was observed in cells lacking hamartin from a murine model of Tsc1,13Kwiatkowski DJ Zhang H Bandura JL Heiberger KM Glogauer M el-Hashemite N Onda H A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in Tsc1 null cells.Hum Mol Genet. 2002; 11: 525-534Crossref PubMed Scopus (547) Google Scholar and in cells lacking tuberin from the Eker rat model of Tsc2,12Goncharova EA Goncharov DA Eszterhas A Hunter DS Glassberg MK Yeung RS Walker CL Noonan D Kwiatkowski DJ Chou MM Panettieri Jr, RA Krymskaya VP Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation: a role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis (LAM).J Biol Chem. 2002; 277: 30958-30967Crossref PubMed Scopus (378) Google Scholar suggesting that the tuberin-hamartin complex negatively regulates S6K. S6K is a critical component of the tightly regulated signal transduction pathways controlling cell size and integrating the external availability of nutrients with protein synthesis (reviewed in27Kozma SC Thomas G Regulation of cell size in growth, development, and human disease: pI3K, PKB, and S6K.Bioessays. 2002; 24: 65-71Crossref PubMed Scopus (257) Google Scholar, 28Blume-Jensen P Hunter T Oncogenic kinase signalling.Nature. 2001; 411: 355-365Crossref PubMed Scopus (3194) Google Scholar, 29Shah OJ Anthony JC Kimball SR Jefferson LS 4E-BP1 and S6K1: translational integration sites for nutritional and hormonal information in muscle.Am J Physiol. 2000; 279: E715-E729Google Scholar). We found that all of the components of the angiomyolipomas in which LOH was detected were immunoreactive with an antibody to phospho-S6. In contrast, the components in which LOH was not detected, including endothelial cells, were not immunoreactive. This is consistent with a model in which hyperphosphorylation of S6 is involved in the pathogenesis of vascular structures within angiomyolipomas. Signaling through S6K is believed to be involved in the proliferation and migration of human vascular smooth muscle cells, and the structural remodeling of vessel walls.30Goncharova EA Ammit AJ Irani C Carroll RG Eszterhas AJ Panettieri RA Krymskaya VP PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells.Am J Physiol. 2002; 283: L354-L363Google Scholar, 31Braun-Dullaeus RC Mann MJ Seay U Zhang L von Der Leyen HE Morris RE Dzau VJ Cell cycle protein expression in vascular smooth muscle cells in vitro and in vivo is regulated through phosphatidylinositol 3-kinase and mammalian target of rapamycin.Arterioscler Thromb Vasc Biol. 2001; 21: 1152-1158Crossref PubMed Scopus (132) Google Scholar, 32Eguchi S Iwasaki H Ueno H Frank GD Motley ED Eguchi K Marumo F Hirata Y Inagami T Intracellular signaling of angiotensin II-induced p70 S6 kinase phosphorylation at Ser(411) in vascular smooth muscle cells: possible requirement of epidermal growth factor receptor, Ras, extracellular signal-regulated kinase, and Akt.J Biol Chem. 1999; 274: 36843-36851Crossref PubMed Scopus (164) Google Scholar In summary, we found that angiomyolipomas contain multiple vessel types, four of which (cellular, hemangiopericytic, glomeruloid, and aneurysmatic) have LOH. One vessel type (collagenous) does not have LOH. The presence of LOH in all three components of angiomyolipomas (vessels, fat, and smooth muscle) supports the hypothesis that angiomyolipomas arise from a mesenchymal precursor cell that retains differentiation plasticity. This distinguishes angiomyolipomas from other benign blood vessel-filled tumors such as those in von Hippel Lindau disease, in which the stromal cells are neoplastic and the vascular cells are not. The presence of hyperphosphorylated ribosomal protein S6 within the neoplastic components of the angiomyolipomas suggests that signaling pathways involving S6K contribute to the formation of the vascular structures within angiomyolipomas. We thank Drs. Andres Klein-Szanto and Andrew Godwin for critical review of this manuscript, and Dr. Al Knudson for many inspiring discussions.