Lymphangiogenesis in rat asthma model

Abstract

Although bronchial angiogenesis has been well documented in allergic asthma, lymphangiogenesis has not been widely studied. Therefore, we evaluated changes in lung lymphatics in a rat model of allergen-induced asthma using house dust mite (Der p 1; 100 μg/challenge). Additionally, properties of isolated lung lymphatic endothelial cells (CD45^-, CD141⁺, LYVE-1⁺, Prox-1⁺) were studied in vitro. Three weeks after the onset of intranasal allergen exposure (twice-weekly), an increase in the number of lung lymphatic vessels was measured (34% increase) by lung morphometry. New lymphatic structures were seen predominantly in the peribronchial and periarterial interstitial space but also surrounding large airways. Isolated lymphatic endothelial cells from sensitized lungs showed enhanced proliferation (% Ki67⁺), chemotaxis, and tube formation (number and length) compared to lymphatic endothelial cells isolated from naive rat lungs. This hyper-proliferative lymphangiogenic phenotype was preserved through multiple cell passages (2-8). Lymphatic endothelial cells isolated from naive and HDM-sensitized rats produced similar in vitro levels of VEGF-C, VEGF-D, and VEGFR3 protein, each recognized as critical lymphangiogenic factors. Inhibition with anti-VEGFR (axitinib, 0.1 μM) blocked proliferation and chemotaxis. Results suggest that in vivo sensitization causes fundamental changes to lymphatic endothelium, which are retained in vitro, and may relate to VEGFR downstream signaling.

Keywords: Allergen; Angiogenesis; House dust mite; Lung; Lymphatic vessels.

Figure 1. Representative example showing FACS verification of lymphatic endothelial cells

Cells for in vitro culture were positively selected with Lycopersicon esculentum agglutinin (endothelial cells), and LYVE-1 (lymphatic endothelial cells) during the proliferation phase. Cells were characterized by flow cytometry after digestion and staining. In this representative example, 94.2% of cells were LIVE cells. Of that population, 97% were endothelial cells (CD141⁺) and not leukocytes (CD45⁻). Of this population 94.2% were Prox-1⁺, a unique intracellular lymphatic endothelial cell marker. This example was from 8^th passage lymphatic endothelial cells.

Figure 2. H&E stained histologic sections of the bronchovascular interstitial space from control (PBS) and HDM exposed rats

In the HDM exposed rats, bronchial-associated lymphoid tissue is marked by the mass of black staining cells located adjacent to increased numbers of small bronchial arteries (red arrows). Enlarged lymphatic vessels (green arrows) are also found in this space with HDM exposure compared to control. Bar = 100 μm

Figure 3A. Cross sections of frozen lung from control (PBS) and HDM rats

Green staining is anti-LYVE-1 (lymphatics), red is Griffonia simplicifolia lectin (pulmonary artery; PA), and blue is DAPI (all cell nuclei). White arrows indicate lymphatic vessels. In the control rats, lymphatics are seen in the interstitial space between airways and pulmonary arteries, whereas in lungs from HDM sensitized rats, additional lymphatics appear all around the airway wall. Bar = 50 μm

Figure 3B. Confocal images from naïve and HDM rats

White arrows in left panels indicate region of enhanced magnification in right panels. Arrows in high magnification panels on right indicate sparse anti-LYVE-1 lymphatic endothelial staining (green) in the interstitial area between a pulmonary artery (PA; red: Griffonia simplicifolia lectin) and an airway outlined by DAPI (blue). The HDM section shows more abundant anti-LYVE-1 lymphatic endothelial staining. Left panel bars = 100 μm; Right panel bars= 50 μm

Figure 4A. Fixed lung sections used for lymphatic quantification

Red, chromagen stained LYVE-1⁺ vessels (arrows) are sparse in naïve airways (upper images) compared to HDM lung (lower panels). Right panels are increased magnification of left panel regions indicated by arrows. Bar = 100 μm

Figure 4B. Quantification of lymphatic vessel number in naïve and HDM lungs

A significant increase in the number of LYVE-1⁺ lymphatic vessels was observed in HDM treated rats compared to naïve rats. Each point represents the number (#) of lymphatic vessels from one rat (n=3 rats/group). * indicates p=0.036

Figure 5A. In vitro LEC proliferation (Ki67⁺) of cells isolated from naïve versus HDM exposed rats

Lymphatic endothelial cell isolated from HDM rats and cultured in 2% serum as well as 20% serum, demonstrated significantly enhanced proliferation compared to lymphatic endothelial cells isolated from naïve rats. * indicates p=0.02 vs naive

Figure 5B. In vitro LEC chemotaxis of cells isolated from naïve versus HDM exposed rats

Chemotaxis (cell #/ field) of HDM lymphatic endothelial cells was markedly greater than naïve lymphatic endothelial cells. * indicates p=0.003

Figure 5C. Example of tube formation of LEC isolated from naïve versus HDM exposed rats

Bar = 50 μm

Figure 5D. Quantification of tube formation

Both the sum (Σ) of all tube lengths/high power field and the number of tubes/well were significantly greater in HDM lymphatic endothelial cells compared to those from than naïve rats. * indicates p≤0.05

Figure 6A. VEGF-C and VEGF-D protein in cultured LEC

The levels of VEGF-C and VEGF-D protein in cell pellets as well as secreted in culture media are summed for naïve and HDM lymphatic endothelial cells normalized for total cell protein (pg/mg protein). No difference in VEGF-C or VEGF-D was seen between naïve and lymphatic endothelial cells.

Figure 6B. VEGFR3 protein in cultured LEC

No differences were observed in VEGFR3 normalized to GAPDH in naïve and HDM lymphatic endothelial cells.

Figure 7A. Effects of anti-VEGF receptor blockade on LEC proliferation

Axitinib, a VEGFR1-3 receptor antagonist, had a significant anti-proliferative effect on both naïve and HDM lymphatic endothelial cells and eliminated the differences observed in HDM-induced proliferation at both inhibitor concentrations. *p<0.05 vs naïve vehicle

Figure 7B. Effects of anti-VEGF receptor blockade on LEC chemotaxis

Axitinib had a significant effect on cell chemotaxis in both naïve and HDM lymphatic endothelial cells and eliminated the differences observed in HDM-induced proliferation. *p<0.05 vs naïve vehicle, # p<0.05 vs HDM