| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Guest Access | Sign In via User Name/Password
|
|
|
|||||||
Guest Access | Sign In via User Name/Password |
|||||||||
* From the Departments of Pathology (Drs. Assaad and Borczuk), Medicine (Drs. Kawut, Arcasoy, and Wilt), Pediatrics (Dr. Rosenzweig), and Surgery (Dr. Sonett), Columbia University College of Physicians and Surgeons, New York, NY.
Correspondence to: Alain C. Borczuk, MD, Department of Pathology, Division of Surgical Pathology, 630 West 168th St, VC14–215, New York, NY, 10032; e-mail: ab748{at}columbia.edu
Abstract
Background: Pulmonary capillary hemangiomatosis (PCH) is a rare cause of pulmonary arterial hypertension with no effective medical therapy and a high risk of mortality. The pathogenesis of PCH is unknown.
Methods: We used gene expression analysis to compare lung tissue samples from two patients with PCH to those from seven control subjects. The nodules of proliferating capillaries in PCH patients were needle microdissected from cryostat sections. RNA extraction and labeling were followed by hybridization to U95Av2 oligonucleotide arrays (Affymetrix; Santa Clara, CA). In situ hybridization and immunohistochemistry were also performed.
Results: The gene expression profile of PCH allowed for unsupervised clustering from the profile of the lung tissue samples of control subjects. Platelet-derived growth factor (PDGF)-B gene (PDGFB), PDGF receptor (PDGFR)-ß gene (PDGFR-ß), mast cell-related genes, and type 2 pneumocyte-related genes were found to be overexpressed in PCH lesions. In situ hybridization as well as immunohistochemistry for PDGFB showed expression by type 2 pneumocytes and endothelial cells. Immunohistochemical staining for PDGFR-ß localized to pericytic/vascular smooth muscle cells surrounding the proliferating capillaries. CD117 staining confirmed an abundance of mast cells in the lesions, which also stained heavily for PDGFR-ß.
Conclusions: The expression of the PDGFB and PDGFR-ß genes characterizes the nodular proliferations of PCH. Increased numbers of mast cells, pericytes, and type II pneumocytes accompany the endothelial proliferation. The up-regulation of these important angiogenic and antiapoptotic genes suggests a mechanism and potential therapeutic approaches for PCH.
Key Words: gene expression profiling • platelet-derived growth factor • pulmonary hypertension • receptor
Pulmonary capillary hemangiomatosis (PCH) is an extraordinarily rare disease that is characterized by abnormal angiogenesis confined to the lung. Wagenvoort et al1 first described PCH in 1978, and fewer than 50 cases have been published since.2 The histopathologic findings of PCH (which has been renamed pulmonary microvasculopathy) include a proliferation of capillary-sized vessels within the alveolar walls, interstitium, and postcapillary venules of the lung.3 Intimal thickening and medial hypertrophy of the small muscular pulmonary arteries are present, which is similar to the findings for other types of pulmonary arterial hypertension (PAH), resulting in elevated pulmonary vascular resistance.34 The pathogenesis of PCH is unknown, and there are no effective medical therapies. Lung transplantation is curative; however, due to the lack of awareness of and difficulty in making this diagnosis, the majority of reported cases have been discovered postmortem.
The similarity between the changes in the small muscular pulmonary arteries in patients with PCH and those changes found in patients with other, more common forms of PAH means that molecular discoveries in the former condition may enlighten the understanding of the latter. Finding the characteristic protein expression shared between PCH and other forms of PAH would suggest that targeted therapies might be effective in patients with all forms of PAH. However, few studies have investigated the mechanism of PCH.5678 With so little known about PCH, high-throughput gene expression profiling may be an efficient way to elucidate potentially important pathophysiologic pathways in patients with this cryptic condition. We therefore assessed the gene expression profiles of microdissected lung nodules obtained from patients with PCH and compared the findings to those of normal lung tissue. These data have been previously presented in abstract form.9
Materials and Methods
We obtained lung tissue samples from two patients with PCH and normal lung tissue from seven patients who had undergone lobectomies performed for neoplastic disease. These control subjects were similar to those in the single published microarray study of PAH.10
Case 1
A 23-year-old woman presented with dyspnea and was found to have severe pulmonary hypertension with a normal pulmonary capillary wedge pressure. There was no family history of pulmonary hypertension. A CT scan of the chest showed nodular opacities and ground-glass infiltrates. A surgical lung biopsy specimen revealed PCH. Bilateral sequential lung transplantation was performed; the patient continues to have normal exercise tolerance > 4 years after surgery.
Case 2
An 18-year-old man presented with progressive shortness of breath, exercise intolerance, clubbing, and cyanosis. Evaluation revealed severe pulmonary hypertension and abnormal findings on a CT scan of the chest. There was no family history of pulmonary hypertension. A surgical lung biopsy specimen showed PCH. Despite treatment with interferon 
-2a, his clinical condition worsened, and he underwent bilateral sequential lung transplantation. He died > 1 year after transplantation due to complications of bronchiolitis obliterans syndrome.
Microarray Hybridization, In Situ Hybridization, and Immunohistochemistry
Tissue specimens from two different lobes of the explanted lungs (case 1) and from both the biopsy and explanted lungs (case 2) were snap-frozen. PCH nodules were microdissected with a 20-gauge needle under light microscopy and were collected into guanidine thiocyanate for RNA extraction (RNeasy Mini Kit; Qiagen; Valencia, CA). After examination of the frozen section, serial sections of the control lung were homogenized and extracted for RNA. Comparing PCH nodules to control lung homogenates avoided assumptions regarding the importance of certain cells or regions of the control lung tissue. PCH nodules were analyzed (as opposed to using cell-specific laser capture) in order to characterize the milieu of the cellular microenvironment of PCH, rather than the genetic expression of specific cell types in the absence of evidence that certain cells have particular mechanistic importance in PCH.
See the "online Appendix" (available at www.chestnet.org) for complete details of laboratory methods. Briefly, target complimentary RNA was hybridized to the U95Av2 oligonucleotide array (Affymetrix; Santa Clara, CA). For the purposes of all subsequent analyses, the second sample from each PCH patient was treated as a replicate; the lung tissues specimens from the seven control subjects were independent samples. Unsupervised clustering was performed using appropriate software (GeneSpring, version 6.1; Agilent Technologies; Santa Clara, CA). Supervised analysis of genes associated with class assignments was performed using the software GENECLUSTER (available at: http://www.genome.wi.mit.edu/cancer/software/genecluster2/gc2.html). As the PCH samples came from microdissected parenchymal lesions and the control samples consisted of whole-lung homogenates, we only present genes with increased expression in the PCH samples. We chose this strategy because genes with decreased expression in the PCH samples compared to those from control subjects could be attributable to differences in tissue sample composition between those from microdissected lesions (PCH patients) and those from whole lung (control subjects).
Paraffin-embedded tissue sections (5 µm) were used for immunoperoxidase staining on an autostainer (Dako; Carpinteria, CA) using a detection system (Envision plus; Dako) with diamino benzidine. In situ hybridization for the platelet-derived growth factor (PDGF)-B gene (PDGFB) was performed on cryostat sections using a biotinylated probe (Gene Detect North America; Sarasota, FL). This study was approved by the Columbia University Institutional Review Board.
Results
High-power histologic examination showed that the alveolar septa exhibited proliferations of capillary-sized vessels on both sides of the alveolar wall, giving a nodular appearance (Fig 1 ). Small and large muscular pulmonary arteries showed hypertensive changes, including intimal and medial thickening without plexiform lesions.
|
Immunohistochemistry was performed to validate the gene expression findings. CD31 and CD34 immunohistochemistry was positive in the endothelial cells of the proliferating capillaries (Fig 2
, top, A). Perivascular/myoid cells were positive for -smooth muscle actin (SMA) [Fig 2, middle top, B] and were negative for desmin. Staining for thyroid transcription factor-1 highlighted type II pneumocyte hyperplasia within the PCH nodules (Fig 2, middle bottom, C). CD117 staining showed the presence of mast cells diffusely throughout the lung parenchyma with high numbers within the nodules (Fig 2, bottom, D).
|
|
Discussion
PCH has been reclassified as a form of PAH associated with significant venous or capillary involvement.11 PCH has a peak incidence in the second and third decade, affecting men and women equally.2 Most cases appear sporadic, with one report of familial occurrence.12 Imaging with chest radiography and CT scanning shows nodular pulmonary infiltrates, septal lines, lymphadenopathy, and occasional pleural effusions, distinguishing PCH from other forms of PAH1314; however, a definitive diagnosis can be made only by histologic examination. Investigators have reported515 successful outcomes using interferon -2a and doxycycline in patients with PCH without significant pulmonary hypertension, although anecdotal experience with these treatments has been disappointing, as was the case with our second patient.
This is the first high-throughput investigation of the molecular pathogenesis of PCH. Using this hypothesis-generating technique, we have shown an increased PDGFB gene message and PDGF-ß protein expression in the small muscular pulmonary arterial and capillary endothelium, and type 2 pneumocytes in PCH patients. PDGFR-ß was expressed by the large number of and pericytes that were present in the micronodules. The only other microarray study10 of lung tissue in patients with other, more common forms of PAH did not share any of the genetic expression signature seen in the PCH patients in our study.
PDGF is a heterodimeric molecule that is composed of two similar polypeptides (A and B) that acts as a potent mitogen for pulmonary artery smooth muscle cells via its tyrosine kinase receptors. PDGF-B has a central role in angiogenesis and in the recruitment of pericytes into newly formed blood vessels; PDGF-B-deficient mice are characterized by the absence of pericytes and vascular microaneurysms.16 The presence of PDGF-B and PDGFR-ß within lesions in PCH patients points to a paracrine loop that stimulates endothelial proliferation and pericyte recruitment and inhibits apoptosis.16171819 The up-regulation of PDGF-B by type II pneumocytes in PCH patients is similar to that seen in patients with other interstitial lung diseases.1718 Data show that bone morphogenetic protein normally inhibits PDGF-induced DNA synthesis, suggesting a link between these second messenger pathways.20
Recent studies212223 of hypoxic and monocrotaline animal models have shown the importance of PDGFR-ß in the development of pulmonary hypertension. One study22 demonstrated the reversal of pulmonary hypertension in these animal models with imatinib mesylate (Gleevec; Novartis; Basel, Switzerland), which is a tyrosine kinase inhibitor that binds PDGFR and is currently used to treat chronic myelogenous leukemia. This study also found increased PDGFR-ß protein expression in whole-lung homogenates obtained from four patients with PAH. There have been several recent case reports242526 suggesting the successful treatment of PAH with imatinib mesylate. Considering the prominent expression of PDGF-B and PDGFR-ß in PCH patients, this proliferative pathway may be a common link between the vasculopathy seen in PCH patients and that seen in patients with other forms of PAH. Antiproliferative therapies may therefore be useful in this currently untreatable disease.
The documentation of the prominent pericytic component in the nodules of PCH patients is also novel. The structural role of the pericyte during angiogenesis may suggest targeted therapeutic approaches to induce the regression of capillary ingrowth.27 An increased number of mast cells was seen in our patients with PCH and has been documented in other forms of pulmonary hypertension in humans.282930 Mast cell granules contain potent proangiogenic substances.31 Tryptase acts directly on endothelial cells to form tube-like structures, degrades the extracellular matrix, and permits capillary invasion into the interstitium.32 In addition, mast cell tryptase expression dramatically increases in the rat monocrotaline model of pulmonary hypertension.33 The number of tryptase-expressing mast cells in lung tumors is associated with vascular density and tumor progression, whereas tumors in mast cell-deficient animals show decreased angiogenesis.3435
There are some limitations to our study. We studied a small number of patients; however, there have been fewer than 50 cases of PCH reported in the entire published literature, making our gene expression analysis a rare opportunity to understand this cryptic disease. Certainly, these findings should be verified in future cases. Increased gene expression and the presence of certain cells do not necessarily prove a causal role, as these findings could just reflect epiphenomena of another primary process. In addition, gene expression analysis should be considered to be hypothesis-generating due to the chance of type I error. Reassuringly though, our findings correspond to recent animal and human studies of pulmonary hypertension, increasing the likelihood of true mechanistic roles for the identified cytokines and cell types. The laser capture microdissection of cells present both in PCH patients and in healthy subjects could provide further insight into the disease process.
In conclusion, PCH is a rare form of pulmonary hypertension characterized by disordered angiogenesis. PDGF-B, PDGFR-ß, and mast cell proteins may play a role in PCH pathogenesis. Increased awareness of this disorder and further studies of these candidate proteins may better elucidate the mechanism of disease and suggest novel therapies for this currently untreatable illness.
Footnotes
Abbreviations: PAH = pulmonary arterial hypertension; PCH = pulmonary capillary hemangiomatosis; PDGF = platelet-derived growth factor; PDGFR = platelet-derived growth factor receptor; SMA = smooth muscle actin
This research was supported in part by the Department of Pathology, Columbia University College of Physicians and Surgeons, and National Institutes of Health grant HL67771.
The authors have reported to the ACCP that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.
Received for publication July 5, 2006. Accepted for publication October 3, 2006.
References
-2a. N Engl J Med 1989;320,1197-1200[ISI][Medline], interleukin-6, platelet-derived growth factor-B and granulocyte-macrophage colony-stimulating factor mRNA in cells of bronchoalveolar lavage fluids from patients with sarcoidosis. Sarcoidosis Vasc Diffuse Lung Dis 1996;13,139-145[ISI][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
