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Background: Pulmonary arterial hypertension (PAH) is a progressive lung disease of unknown etiology characterized by increased pulmonary artery pressure with obstructive changes in lung vascular structure. Currently the role of pulmonary lymphatics in PAH and whether lymphatic reorganization takes place during PAH is not known. Therefore, we investigated lymphatic remodeling in the pathophysiology of human PAH.
Methods and Results: By H&E staining and immunostaining of archival paraffin sections of control and PAH-derived lung specimens for the lymphatic specific marker D2-40 (podoplanin). A comparison of lymphatic vessel numbers and sizes in these specimens showed an overall decrease in the number of lymphatics (p=0.023) but an increase in the size of these vessels (p=0.0576). We also examined how inflammation-induced lymphatic dysregulation was influenced by cytokines. Because lymphatic endothelial VEGF receptor-3 controls lymphatic abundance and integrity we examined its expression in response to interferon-Î³ (IFN-Î³), interleukin-1Î² (IL-1Î²) and tumor necrosis factor-Î± (TNF-Î±). Our results show that IL-1Î² decreased VEGFR-3 at all doses.
Conclusions: These findings suggest that patients with PAH may have fewer, but larger lymphatics which contribute to disease and hypothesize that accumulated immune cells and cytokines may provoke this remodeling via dysregulation of lymphangiogenic receptors.
Pulmonary arterial hypertension is a disease identified by extensive vascular remodeling, which over time causes a gradual increase in pulmonary arterial pressure and pulmonary vascular resistance. The remodeling, which is promoted by fibroblast proliferation, inflammatory mechanisms, and the activation of cytokines , is characterized by both varying degrees of luminal narrowing, due to endothelial cell proliferation , as well as increased production and deposition of extracellular matrix . It is a lethal disease often leading to right ventricular heart failure .
PAH can develop idiopathically, or in association with several diseases including collagen vascular disease, valvular heart disease, scleroderma, systemic lupus erythematosus, portal hypertension, human immunodeficiency virus infection, use of the combination anorexigens Fenfluramine-Phentermine, drug use (e.g. amphetamines and cocaine) and hypoxia secondary to obstructive sleep apnea or obesity hypoventilation syndrome . In addition, a small percentage of the individuals with PAH appear to acquire it genetically with this syndrome, behaving as an autosomal dominant disorder with incomplete penetrance and genetic anticipation .
In PAH, pulmonary artery pressure increases when small pulmonary arteries become abnormally stenosed or blocked.Â Three pathological mechanisms have been proposed to explain this increase in pulmonary artery narrowing in PAH: 1) arteriolar smooth muscles may contract excessively, increasing resistance to blood flow ; 2) microemboli may obstruct the smaller arteries, leading to increased flow resistance  and 3) arteriolar walls may become hyperplastic/hypertrophic (thickened) from inappropriate or exuberant vascular cell growth (smooth muscle and endothelial cells) induced by mediators e.g. Endothelin-1, serotonin, thromboxane A2, vascular growth factors, and reduction of anti-proliferative mediators, such as nitric oxide and prostacyclin.
Several studies have shown that inflammation plays a prominent role in human PAH and elevated levels of several pro-inflammatory cytokines, e.g. interleukin-1ï¢ (IL-1ï¢) and IL-6, as well as an increased pulmonary expression of macrophage inflammatory protein-1a (MIP-1a) are found in PAH. Further evidence supporting the concept of a systemic inflammatory component in PPH was provided by Balabanian et al., who showed that patients with severe PPH had an increase in the plasma levels of inflammatory markers, compared with normal controls .
Cytokines are known to play important roles in inflammation by activating endothelial cells to mobilize adhesion molecules , entrap leukocytes  and contribute to regulation of immune responses . Interleukin-1Î² (IL-1Î²), interferon-Î³ (IFN-Î³), and tumor necrosis factor-Î± (TNF-Î±) are prominent among the numerous cytokines known to play a pivotal role in inflammation. TNF-Î± also mediates prostaglandin and interleukin-1 synthesis , and activates endothelial adhesion molecule expression [13, 26]. IL-1Î² increases prostaglandin synthesis , which may promote to vasoconstriction. IFN-Î³ stimulates monocytes/macrophages , and modulates ICAM-1 expression , and is strongly implicated in the pathogenesis of several autoimmune and chronic inflammatory diseases .
We have previously shown that IFN-Î³, Il-1Î², and TNF-Î± reduce LEC proliferation, induce ECAM surface expression, and decrease capillary integrity . Their ability to reduce capillary formation in particular, was interpreted as evidence that the cytokines are anti-lymphangiogenic. Patients with PAH have been shown to have significantly elevated levels of IL-1Î², TNF-Î±, and several other plasma cytokines .
Pulmonary lymphatics play a central role in transport of pulmonary interstitial fluid, immune cells and importantly, inflammatory cytokines out of the lung . Therefore, disturbances in pulmonary lymphatics might lead to reduced clearance of these mediators leading to their accumulation and elevation in PAH. These products have been shown to be clearly toxic in studies of 'shock lymph' ;. Lymph from inflamed or injured lung was found to contain high levels of many inflammatory mediators which contribute to systemic injury ; when diverted from the circulation, systemic injury is prevented. Clearly, maintenance of these products in the lung due to altered lymphatic function or density might lead to intense local lung injury which could contribute to the etiology of PAH. It is still controversial whether lymphatics are increased, decreased or exhibit altered function in PAH, but could significantly impact our understanding of its underlying mechanisms and treatments.
In order to investigate whether how lymphatics are altered in human PAH and how they might be affected by cytokines, we measure the expression of D2-40, a monoclonal antibody that stains the endothelium of lymphatic vessels, in normal lung endothelium as compared to the lung endothelium of patients diagnosed with pulmonary arterial hypertension. This comparison could discern a correlation between lung lymphatics and pulmonary arterial hypertension. The role of cytokines on the expression of lymphatic vascular endothelial growth factor receptor-3 (VEGFR-3) expression, a receptor uniquely exclusive and commonly expressed in lymphatic endothelial cells  was also studied. Our data provide support for the concept that PAH injury may reflect obliterative or obstructive remodeling of lung lymphatic, which could be modulated by cytokine effects on lymphatic VEGFR-3 mediated lymphatic survival.
Materials and Methods
Lung tissue from normal individuals (n=4) and patients with idiopathic pulmonary arterial hypertension (n=5) exhibiting classic plexiform lesions were obtained through bronchoscopy with transbronchial biopsy. Samples came from the Program in Translational Lung Research, University of Colorado Health Sciences Center (Denver, Colorado) and Louisiana State University Health Sciences Center (Shreveport, Louisiana) through tissue banks maintained by their departments of pathology, in accordance with the IRB regulations of the appropriate institutions. It is to be noted that the patients, from which the samples were collected, were all diagnosed with systemic sclerosis (SSc), a chronic autoimmune connective tissue disease. PAH occurs in roughly one in seven patients with SSc . Samples were immediately fixed in formalin and paraffin embedded until processed for immunochemical staining.
Preparation of Samples
Paraffin embedded lung tissue sections were prepared for immunohistochemical staining for human podoplanin. Podoplanin is a transmembrane mucoprotein (38 kD), a lymphatic endothelial marker recognized by the D2-40 monoclonal antibody (Covance, Emeryville, California). Podoplanin is specifically expressed in the endothelium of lymphatic capillaries but not in the blood vasculature [7, 21]. The tissue sections were stained using 3% H2O2/methanol (30Â°), to reduce non-specific background staining, followed by staining with 1:250 anti-human podoplanin (D2-40), and visualizing the stain by horseradish peroxide (HRP) and 3,3'-diaminobenzidine (DAB). The primary antibody, a goat anti-mouse Ig HRP conjugate was fixed onto the D2-40 mouse anti-human podoplanin.
After staining of the slides they were analyzed under bright field microscopy. A digital Olympus BX50F light microscope (Olympus Optical Co. Ltd., Japan) was used to view the lung tissue slides at 40x and 100x magnification. Images were captured using One-TouchTM video capture (Diamond Multimedia, Los Angeles, CA). At least ten random images were photographed from each slide for evaluation of lymphatic vessel numbers and surface area. Images were saved to computer and further analysis for lymphatics is performed using Image-J software. Lung vasculature was distinguished based on size, lining of endothelial cells, and specific characteristics of arteries and veins.
Culturing Lymphatic Endothelial Cells
Murine lymphatic endothelial cells (LEC) were cultured as described by Ando et. al. . The cells were subsequently subjected to treatment with various doses of the three cytokines (n=4).
Treatment with Cytokines
Interferon- Î³ (IFN-Î³) was given at doses between 0 and 1000 ng/ml; interleukin-1Î² (Il-1Î²) was given at doses between 0 and 10 ng/ml; and tumor necrosis factor- Î± (TNF-Î±) was given at doses between 0 and 20 ng/ml. All assays were performed (n=4) for each cytokine testing the various doses over a 72 hour time-period.
A cell surface binding cell-based ELISA assay was administered on a 96-well plate. Samples of IFN-Î³, Il-1Î², and TNF-Î± were prepared according to dose and time, and then 100 Î¼l were pipetted into the labeled wells on the plate, followed by cell fixation with formalin. The wells, containing LECs and cytokines, were then reacted with anti-VEGFR-3 for 1 hour and washed with buffer, followed by a second reaction with anti-rat-HRP and wash with buffer. Tetramethylbenzidine/peroxide (TMB/H2O2) was added to detect color, followed by the addition of 8M sulfuric acid to stop the color reaction. The subsequent absorbances were measured using a spectrophotometer.
Data and Statistical Analysis
Image J software (NIH) was used to evaluate the number of lymphatic vessels and surface area of lymphatic vessels. All the images were analyzed using a tablet PC and Image J software. Lymphatic vessels were clearly outlined and surface area calculated using built in functions of Image J software. At the same time, surface area of the tissue is analyzed as well. The lymphatic vessels surrounding the arteries and veins, stained a dark brown due to D2-40+ immunostaining, and were manually accounted for under the microscope and analyzed using Image-J with pixel-to-distance correction. Statistical analysis was carried out using GraphPad InStat (GraphPad Software, Inc., La Jolla, California). Graphics were prepared using the software SigmaPlot 2001 (SPSS Inc., Chicago, Illinois).
Specificity of Staining
The specificity of our staining shows discrimination between the vascular immunohistochemical (IHC) marker CD31/PECAM-1 staining blood vessels and the lymphatic IHC marker D2-40 staining lung lymphatics. CD31 stains the blood vessels a light brown color, while the D2-40 stain the lymphatics a dark brown (Figure 1). A digitalized Olympus CK2 light microscope was used to view the lung tissue slides at 40x and 100x magnification. Lung vasculature was distinguished based on size, lining of endothelial cells, and specific characteristics of arteries and veins. The lymphatic vessels surrounding the arteries and veins, stained a dark brown, were manually accounted for and analyzed using Image-J with pixel-to-distance correction (Figure 2).
Perivascular lymphatic vessels
The arteries overall appear to be surrounded by more lymphatic vessels than the venules. However, the PAH group had slightly reduced numbers of perivascular lymphatic vessels surrounding arteries (but not statistically significant). There was also no difference in lymphatic vessel numbers surrounding veins between PAH and controls. In the PAH group, there was an average of 7.3 Â± 1.5 lymphatic vessels surrounding the arteries and an average of 5.18 Â± 0.5 lymphatic vessels surrounding the veins (p=0.23). In the normal group, there was an average of 10.18 Â± 2.7 lymphatic vessels surrounding the arteries and an average of 5.6 Â± 0.94 lymphatic vessels surrounding the veins (p=0.11).
Number of lymphatic vessels per square millimeter of tissue
The PAH group showed significantly fewer lymphatic vessels per square millimeter than control. The PAH group had 8.9 Â± 2.2 lymphatic vessels per square millimeter of tissue, while the normal group had 16.2 Â± 5.32 lymphatic vessels per square millimeter of tissue (p=0.02) (Figure 3).
Average area of lymphatic vessels
The PAH group had lymphatic vessels with greater average surface areas compared to the controls. The average area of one lymphatic vessel in the PAH group was 0.008 Â± 0.007 Âµm2 (n=4), while in the normal group it was 0.002 Â± 0.001 Âµm2 (n=4, p=0.05) (Figure 4).
Ratio of the area of lymphatics per tissue
PAH group had a greater number of lymphatics per area than the controls. The PAH group had 7.39 Â± 4.56 mm2 (n=4) per tissue mm2, while the normal group had 3.38 Â± 0.98 mm2 per tissue mm2 (n=4, p=0.06) (Figure 5).
Effect of IL-1Î² on VEGFR-3 expression
IL-1Î² dose and time dependently increased VEGFR-3 on expressed on the cell surface, followed by decreased expression at, intensifying at low doses. At 24 hours after exposure to 5ng/ml and 10ng/ml Il-1Î², we found a significant (p<0.05) decrease in VEGFR-3 (47+/-4% and 49+/-4% of control levels, respectively) (Figure 6). At 48 hours after exposure to 1ng/ml Il-1Î², we found a significant (p<0.05) increase in VEGFR-3 (134+/-22% of control levels), followed by a significant (p<0.05) decrease in VEGFR-3 (33+/-7% and 30+/-6% of control levels) at 5ng/ml and 10ng/ml Il-1Î², respectively. However, neither of the above concentrations had any significant effect at 72 hours.
Effects of TNF-Î± and IFN-Î³ on VEGFR-3 expression
TNF-Î±, tested at 5, 10, and 20ng/ml, was found to be too statistically variable over the 72 hour time period to significantly conclude any effect on the surface expressed VEGFR-3 (Figure 7). IFN-Î³ was found to increase the surface expression of VEGFR-3 in a dose and time-dependent manner at high doses. At 48 and 72 hours, after exposure to 1000U/ml IFN-Î³, we found a significant (p<0.05) increase in VEGFR-3 (165+/-35% and 148+/-23% of control levels, respectively) (Figures 8).
In this study, we examined the role of the pulmonary lymphatic vasculature in patients with pulmonary arterial hypertension. The effects of inflammatory cytokines were also examined to further understand their role in lymphatics, and thus their function in pulmonary arterial hypertension.
Lymphangiogenesis, the formation of lymphatic vessels from pre-existing lymphatic vessels or recruitment of circulating progenitors is a phenomenon that plays a role in homeostasis and immunity, and has been observed in numerous inflammatory and autoimmune disorders. With regards to vascular proliferation and remodeling, lymphangiogenesis differs from its counterpart, angiogenesis, the process involved in the growth of new blood vessels. During inflammatory diseases, blood vessels quickly return to their normal state as the inflammation subsides through restitution of the vessels . However, lymphangiogenesis appears to reverse more slowly and the persistence of the newly formed lymphatic network even after the inflammation is resolved, may leave a "drainage system" in place for fluid and immune cells. Potentially this could be beneficial by helping to maintain lymphatic drainage from the lungs, or deleterious by accelerating the responses in future inflammatory occurrences .
Lymphangiogenesis is closely tied to inflammation through the production/release of VEGF-C and VEGF-D, growth factors capable of inducing lymphangiogenesis through VEGFR-3 signaling mechanisms . This may help control edema by clearing lung interstitial fluid, but may enhance immune trafficking, leading to heightened immune responses. In addition to VEGF-C and VEGF-D, VEGF-A plays a role in lymphangiogenesis by stimulating lymphatic growth through the recruitment of monocytes/macrophages, which release VEGF-C/VEGF-D .
Studies and treatment methods for PAH have yet to place any significant focus on the issue of lymphatic dysfunction. Considering one of the common factors between angiogenesis and lymphangiogenesis is the family of VEGF, a treatment regimen designed around increasing this growth factor could potentially alleviate the problems associated with the decreased amount of lymphatics. For example, Baicilin induces VEGF expression through the activation of the ERRÎ± pathway  and produces evidence of oxidative stress (peroxynitrite) through activation of STAT3 . Coupled with vasoactive medications, this approach could allow for the proper channeling of blood flow as well as a manner in which the excess fluid could be removed and pressure to be decreased.
It is unclear whether VEGFR-3, the lymphangiogenic-promoting receptor tyrosine kinase, is down-regulated in pulmonary arterial hypertension as it is in lymphedema [35, 41]. Blockade or reduction of lymphatic VEGFR-3 has been suggested as a therapeutic strategy for inhibiting lymphatic outflow which suppresses tumor metastasis . With respect to cultured murine lymphatic endothelial cells (LECs), evidence from our lab also indicates that VEGFR-3 is down-regulates under inflammatory conditions. Using a cell surface binding ELISA assay, VEGFR-3 expression was examined for changes in response to increasing doses of interferon-Î³ (IFN-Î³), interleukin-1Î² (Il-1Î²) and tumor necrosis factor-Î± (TNF-Î±).
In agreement with data indicating significantly elevated levels of IL-1Î², among other cytokines in patients with idiopathic and heritable PAH, it could be inferred that such patients might down-regulate VEGFR-3, with an overall reduction in lymphatic density . The significance of VEGFR-3 down-regulation in PAH was also supported by Chaitanya et. al. also showed other forms of lymphatic dysregulation induced in lymphatic endothelium by IL-1Î², TNF-Î± and IFN-g, cytokines present in PAH patients .
With regards to pulmonary arterial hypertension, associations with both angiogenesis and lymphangiogenesis have been noted in previous literature as well as found in this study. Tuder et. al. presented evidence of endothelial cells expressing genes that encode for angiogenesis-related proteins, namely VEGF and VEGFR-2 , as well as immunohistochemical evidence of angiogenesis-related molecules being present in the plexiform lesions of patients with severe pulmonary arterial hypertension . These lesions, a hallmark symptom of severe PAH, are glomeruloid structures composed of proliferated endothelial cells that form within the pulmonary arteries . The evidence overall suggested that pulmonary arterial hypertension occurred through a process of disordered, but increased angiogenesis. This increase allows for additional blood supply to the damaged tissue, and thus for greater efficiency bringing in platelets, leukocytes, and fibroblasts to the particular area.
Schraufnagel et al. demonstrated an increase in lymphangiogenesis in a rat model of pulmonary arterial hypertension. Utilizing scanning electron microscopy of injected plastic casts, that study found that all compartments of the lung lymphatics expanded after the injury and edema caused by hypoxia, and returned to their normal state as the edema subsided . An increase in the lymphatic vasculature around the blood vessels should accommodate the removal of the edema-induced excess fluid, relieving the interstitial pressure in the lungs.
Several important differences exist between Schraufnagel's rodent model of pulmonary arterial hypertension and human PAH. Pulmonary edema, which is an accumulation of fluid in the lungs, is prevalent in the acute rat model. The development of this edema in the experimental rats, after continuous hyperoxic conditions, triggers extensive lymphatic expansion . Although it would be anticipated that pulmonary edema would also be a symptom for chronic human PAH, this is not observed. It is however seen in association with lung infections, pulmonary venous hypertension, and pulmonary veno-occlusive disease , among other causes. Another distinction between the human and rat models of PAH was that the rats were able to recover and return to their pre-existing state, once the hyperoxic conditions were removed and the edema subsided; whereas no current treatment can cure the condition in the chronic human form .
Considering studies by Tuder et al. and Schraufnagel et al., it might be assumed that pulmonary arterial hypertension in humans would show similar behavior, such that there would be a characteristic increase in both angiogenesis and lymphangiogenesis during PAH. Our current study however, focusing on the lymphatic vasculature surrounding the blood vessels, presents evidence of the opposite scenario taking place. That is to say, because lymphatics relieve excess tissue fluid and leukocyte accumulation, the relative deficit and enlargement of this lymphatics seen here in human pulmonary arterial hypertension suggests that lymphatic dysfunction may contribute to the pathophysiology of pulmonary arterial hypertension through inflammatory cell infiltration, angiogenesis, and proliferation of smooth muscle and other adventitial cell types [22, 36].
This study has described new relationships between lymphatics and human pulmonary arterial hypertension, as well as further evidence of these lymphatics being modulated by inflammatory cytokines. These findings may have implications on the relationship between lymphangiogenesis, angiogenesis and inflammation, and may contribute to the development of new, more effective treatment regimens for this devastating disease. Future studies should be aimed at further investigation of growth factors regulating lymphatic vessels, to better understand the contribution it makes on the vascular system in the initiation and progression of pulmonary arterial hypertension.