Lymphatic Endothelial Cell Defects in Congenital Cardiac Patients With Postoperative Chylothorax

Abstract

Objectives: Chylothorax following cardiac surgery for congenital cardiac anomalies is a complication associated with severe morbidities and mortality. We hypothesize that there are intrinsic defects in the lymphatics of congenital cardiac patients.

Methods: Postsurgical chylothorax lymphatic endothelial cells (pcLECs) (n = 10) were isolated from the chylous fluid from congenital cardiac defect patients, and characterized by fluorescent-activated cell sorting, immunofluorescent staining, and quantitative RT-PCR. Results were compared to normal human dermal lymphatic endothelial cells (HdLECs). pcLECs (n = 3) and HdLECs were xenografted into immunocompromised mice. Implants and postoperative chylothorax patient's pulmonary tissues were characterized by immunostaining for lymphatic endothelial proteins.

Results: pcLECs expressed endothelial markers VECADHERIN, CD31, VEGFR2, lymphatic endothelial markers PROX1, podoplanin, VEGFR3, and progenitor endothelial markers CD90 and CD146. However, pcLECs had key differences relative to HdLECs, including altered expression and mislocalization of junctional proteins (VECADHERIN and CD31), and essential endothelial proteins, VEGFR2, VEGFR3, and PROX1. When xenografted in mice, pcLECs formed dilated lymphatic channels with poor cell-cell association. Similar to congenital lymphatic anomalies, the pulmonary lymphatics were dilated in a patient who developed postoperative chylothorax after cardiac surgery.

Conclusions: Recent studies have shown that some postoperative chylothoraces in congenital cardiac anomalies are associated with anatomical lymphatic defects. We found that pcLECs have defects in expression and localization of proteins necessary to maintain lymphatic specification and function. This pcLEC phenotype is similar to that observed in lymphatic endothelial cells from congenital lymphatic anomalies. Co-existence of lymphatic anomalies should be considered as a feature of congenital cardiac anomalies.

Keywords: Chylothorax; Congenital cardiac anomalies; Lymphatic endothelial cells.

Figure 1.

pcLECs expressed endothelial genes. A, Representative HdLECs and pcLECs in culture. B, VECADHERIN, CD31, and VEGFR2 FACS of HdLECs and 3 pcLECs. Blue: specific antibodies, red: IgG control. C, HdLECs and (D) pcLECs immunostained for VEGFR2, PODOPLANIN (PDPN), VECADHERIN (VECAD), and CD31. Scale bars: 50 μm. D, Boxed areas are enlarged to the right. E, TIE2 qRT-PCR of HdLECs and pcLECs normalized to β-actin. Each data point represents a unique cell population (n = 3). *P < .005. Abbreviations: HdLECs, human dermal lymphatic endothelial cells; pcLECs, postsurgical chylothorax lymphatic endothelial cells; qRT-PCR, quantitative RT-PCR.

Figure 2.

pcLECs expressed LEC genes. A, PODOPLANIN and VEGFR3 FACS of HdLECs and 3 pcLECs. Blue: specific antibodies, red: IgG control. C, HdLECs immunostained for PODOPLANIN (PDPN), PROX1, and VEGFR3. pcLECs immunostained for (C) PDPN and VEGFR3 or (D) PDPN and PROX1. B–D, Scale bars: 50 μm. E, PROX1, PODOPLANIN, LYVE1, and VEGFC qRT-PCR of HdLECs and pcLECs normalized to β-actin. Each data point represents a unique cell population (n = 3). *P < .005. Abbreviations: HdLECs, human dermal lymphatic endothelial cells; pcLECs, postsurgical chylothorax lymphatic endothelial cells.

Figure 3.

pcLECs expressed progenitor EC genes. CD90 and CD146 FACS of HdLECs and 3 pcLECs. Blue: specific antibodies, red: IgG control. Abbreviations: HdLECs, human dermal lymphatic endothelial cells; pcLECs, postsurgical chylothorax lymphatic endothelial cells.

Figure 4.

pcLECs form abnormal lymphatic channels in a xenograft mouse model. Five-week HdLECs and pcLEC implants were immunostained for PROX1 and PODOPLANIN. Boxed areas in images in the first column are enlarged to the right. White arrowheads mark lymphatic vessels. Scale bar, 50 μm. Abbreviations: HdLECs, human dermal lymphatic endothelial cells; LC, lymphatic channel; pcLECs, postsurgical chylothorax lymphatic endothelial cells.

Figure 5.

pcLEC-derived lymphatic channels expressed VEGFR2 and VEGFR3. Control HdLECs and pcLECs implants stained for (A) VEGFR2 and PODOPLANIN (PDPN), or (B) VEGFR3 and PDPN. Boxed areas in images in the first column are enlarged to the right. White arrowheads mark lymphatic vessels. Scale bar, 50 μm. Abbreviations: HdLECs, human dermal lymphatic endothelial cells; LC, lymphatic channel; pcLECs, postsurgical chylothorax lymphatic endothelial cells.

Figure 6.

Abnormal lymphatic vasculature in patient with secondary chylothorax. Pulmonary tissues obtained from a patient who developed secondary chylothorax after cardiac surgery (PC tissue) was compared to neonatal dermis (Control). Tissue sections were immunostained for (A) PROX1 and PODOPLANIN (PDPN), (B) VEGFR2 and PDPN, or (C) VEGFR3 and PDPN. Boxed areas in images in the first column are enlarged to the right. White arrowheads mark lymphatic vessels. Scale bar, 50 μm. Abbreviation: LC, lymphatic channel.