THE LYMPHATIC SYSTEM
Lymphatic vessels are required for the absorption of intestinal lipids, transport of immune cells, and return of tissue fluid and macromolecules to the blood vascular system. The hierarchal lymphatic vasculature begins with lymphatic capillaries comprised of a single layer of non-fenestrated endothelial cells connected to the extracellular matrix via anchoring filaments . Once lymph enters lymphatic capillaries, it flows into collecting lymphatic vessels. In contrast to lymphatic capillaries, collecting lymphatic vessels are surrounded by mural cells and contain bicuspid intraluminal valves that partition vessels into discrete contractile elements called lymphangions . The pumping of lymphangions propels lymph through regional lymph nodes to lymphatic-venous junctions . Impaired function of the lymphatic system or an insufficient number of lymphatic vessels can cause the accumulation of fluid and protein in tissues resulting in the debilitating disorder lymphedema. Conversely, new lymphatic vessels form in many pathological settings and promote processes such as the metastasis of cancer cells to lymph nodes and rejection of transplanted tissues . These observations have fueled intense research efforts to identify the molecular mechanisms regulating lymphangiogenesis so that therapies can be developed to promote or inhibit this process to treat human disease.
Gorham-Stout disease (GSD) is a rare disease of unknown etiology characterized by the replacement of bone with fibrous tissue containing numerous endothelial-lined channels . GSD may occur at any age but is most commonly observed in children and young adults . It affects males and females of all races and does not exhibit a clear inheritance pattern . The clinical course of GSD is unpredictable and may rapidly progress or spontaneously arrest . Symptoms are typically nonspecific such as weakness or acute pain and swelling . In some cases, the first symptom is bone fracture following minor trauma . Although GSD can affect any bone in the body, it frequently involves the shoulder, pelvis, jaw, and ribs . Thoracic involvement tends to carry a poor prognosis and can be associated with chylothorax as well as pleural and pericardial effusions [7,9,10,11,12]. These complications can compromise organ function and have dire consequences such as respiratory distress and failure.
Old case reports describing numerous blood vessels in tissue biopsies taken from patients with extensive bone resorption prompted Gorham and Stout to evaluate the histological changes in 8 prior cases of massive osteolysis . In their groundbreaking paper, Gorham and Stout reported the remarkable discovery that angiomatosis is associated with the complete disappearance of bone(s) . They found that in early stages of the disease affected bones contain marrow spaces filled with dilated vessels and that in later stages of the disease bones have been replaced by fibrous tissue littered with endothelial-lined vessels .
The emergence of immunohistochemical markers of the vascular and lymphatic system has greatly facilitated the characterization of the histological changes in GSD. Gorham and Stout suggested that the deranged vessels in pathologic tissue may originate from either the blood vascular or lymphatic system. However, until recently the presence of lymphatic vessels could not be confirmed because of a lack of immunohistochemical markers. LYVE-1 is a receptor for the glycosaminoglycan hyaluronan and is expressed by lymphatic capillaries but is down-regulated by collecting lymphatics. Another marker of lymphatics is podoplanin, a transmembrane glycoprotein recognized by the antibody D2-40 and expressed by lymphatic capillaries and collecting vessels. Importantly, numerous LYVE-1 and podoplanin positive lymphatic vessels are present in GSD lesions [9,14,15]. Vessels in pathological specimens taken from patients with GSD have also been reported to express VEGFR3, CD31, PDGFRβ and endoglin [15,16]. Together, these observations confirm the presence of a lymphatic component in GSD.
The International Society for the Study of Vascular Anomalies (ISSVA) has developed a system to classify anomalies of the vascular and lymphatic system and applied this to GSD. According to the ISSVA classification system, malformations are anomalies which can be distinguished from tumors by the expression of immunohistochemical markers of cell proliferation. Although numerous vessels are present in GSD, the endothelial cells show low a labeling of the proliferation marker MIB-1 [16,17,18] . Therefore, according to the ISSVA system, the anomaly in GSD is a malformation rather than a tumor .
The cause of excessive bone resorption in GSD is unclear. Gorham and Stout maintained that osteolysis could be due to hyperemia, changes in pH, or mechanical forces . Heyden suggested that sluggish blood flow may trigger hypoxia, lower tissue pH and favor the activity of acid hydrolases . Endothelial dysplasia has also been proposed to drive the progression of GSD . More recently, it has been suggested that enhanced osteoclast activity contributes to the pathology of GSD. Osteoclasts are bone degrading cells whose role and even presence in GSD has remained controversial. Gorham and Stout failed to identify osteoclasts in the pathological specimens used in their original investigation. Several other reports also document an absence of osteoclasts in GSD [13,19,21,22,23]. Conversely, osteoclasts have been noted by others [5,24,25]. This inconsistent observation has been proposed to be due to the evaluation of different phases (active or remission) of the disease among the various cases . Despite the inconsistency in the literature concerning their presence, changes in GSD appear to directly affect osteoclasts and their activity. Osteoclast progenitor cells isolated from a GSD patient were found to be more sensitive to the osteoclastogenic factors RANKL and M-CSF . Furthermore, serum from a GSD patient increased the number of osteoclast-like cells compared to normal serum and this activity was blocked by an IL-6 neutralizing antibody . These data suggest that circulating factors could influence osteoclast activity and impact bone resorption in GSD.
There is growing evidence that circulating factors could function as biomarkers to monitor disease activity in GSD. IL-6 is a cytokine involved in bone resorption and has been found to be elevated in two different patients with GSD and to normalize following bisphosphonate treatment alone  or in combination with radiation . However, a recent report questions the use of IL-6 as a biomarker of disease activity because the authors found that the levels of IL-6 did not correlate with the state of the disease . VEGF-A, a ligand of the receptor tyrosine kinases VEGFR1 and VEGFR2 with hem/lymphangiogenic properties [27,28], is also another potential biomarker for GSD. VEGF-A was reported to be elevated in 2 patients with active disease and dropped following disease stabilization . Furthermore, VEGF-A was elevated in the plasma of another GSD patient and normalized with clinical improvement following therapy with interferon alpha 2b . Further exploration of cytokines may lead to the identification of molecules to better monitor the progression and/or remission of GSD in response to therapies.
Currently, there is no standard of care for GSD and most treatments are used to control symptoms. Several different strategies have been employed to treat GSD and exhibit varying degrees of success. Surgical interventions such as pleurodesis and thoracic duct ligation have been widely used to manage the accumulation of fluid in the pleural cavity in GSD patients . Radiation therapy has also been used to control thoracic involvement and disease progression. Hyed et al. , recently reviewed radiation therapy in 44 GSD patients and found disease progression, arrest, and remission occurred in 22.7%, 50%, and 27.3% of patients, respectively.
Therapeutic strategies targeting the deranged endothelium in bones and fibrotic tissue have also been used to treat GSD. Interferon alpha-2b as a single agent exerted a noticeable clinical benefit in a patient with GSD that previously received radiation therapy and ligation of the thoracic duct . Interferon alpha-2b combined with clondronate also showed a therapeutic benefit; however, since both agents were given simultaneously, it is unclear which drug was more important for the improvement in the patient’s condition . Clinical improvement has also been noted in cases where pegylated interferon alpha-2b has been combined with zoledronic acid  or surgery . Interferon alpha with steroid therapy also induced disease remission in a patient . The anti-VEGF-A antibody bevacizumab has also been used to successfully treat a case of GSD .
Bisphosphonates are potent inhibitors of osteoclasts and have been widely used to treat osteolytic diseases. This led to their testing in GSD to block osteolysis. Disease stabilization has been noted in cases which used bisphosphonates to treat lesions affecting the hand , jaw , pelvic girdle , and ribs [11,23]. However, disease progression has been observed even in the presence of long-term use of bisphosphonates .
GENERALIZED LYMPHATIC ANOMALY
Generalized lymphatic anomaly (GLA; formerly called lymphangiomatosis) is rare disease of unknown etiology characterized by an increase in the number of lymphatic vessels . GLA does not exhibit a race or gender predilection and is believed to be a congenital disorder due to an error in the development of the lymphatic system. Although GLA can present at any age, it is typically diagnosed in children . This condition may affect a single organ, but commonly involves multiple organs . Thoracic involvement frequently presents with chylous effusions is associated with a poor prognosis and a high mortality rate, especially in children [39,40]. Symptoms of GLA are nonspecific and include wheezing, cough, dyspnea, and chest pain . These nonspecific symptoms cause GLA to be misdiagnosed as asthma in many individuals . However, imaging (MRI and CT) and histological analysis can help assist in the correct diagnosis of GLA.
The histological features of GLA are distinct from other diseases of the lung. GLA specimens contain numerous anastomosing vessels comprised of flattened-endothelial cells that stain positive for factor VIII-related antigen  and D2-40 . These vessels are accompanied by clusters of HMB-45 negative spindle cells present in varying degrees among individuals . These histological characteristics help distinguish GLA from lymphangiectasia (dilatation of lymphatics) and lymphangioleiomyomatosis (abnormal proliferation of HMB-45 positive smooth muscle cells).
There is currently no standard of practice for treating GLA and most treatments are palliative in nature. The beta blocker propranolol has been used to treat a case of GLA and was reported to control the patient’s pleural effusion and normalize plasma VEGF-A levels . Inhibition of the mTOR pathway with Sirolimus has also been used to treat GLA . Other treatments for GLA include surgical resection , pleurodesis , lung transplantation , interferon alpha [47,48], and medium chain triglyceride diet .
Advances in genetics and lymphatic biology are helping elucidate the molecular pathways controlling the growth of lymphatic vessels. However, the mechanisms driving lymphangiogenesis in GSD and GLA remain unclear. It is our hope that future investigations will: 1) shed light on the underlying mechanisms responsible for GSD and GLA, 2) detect biomarkers for monitoring the progression/remission of GSD and GLA, and 3) identify deregulated pathways susceptible to intervention with targeted therapies to improve the clinical outcome of patients.
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