Congenital diaphragmatic hernia (CDH) is a life-threatening congenital disease which occurs approximately 1:2500 life births. CDH is characterized by a defect in the diaphragm, pulmonary hypoplasia and pulmonary hypertension (PH).
The PH associated with CDH is the cause of long term hospitalization and life-long medical treatment. The difficulty of treating CDH lays in the fact that CDH patients respond differently to therapy for PH.
PH is characterized by a thickening of the smooth muscle cells layer within the tunica media of the arterioles, this is a process called hyper-muscularization. The small capillaries in the distal end of the lung are also muscularized, a process what is called neo-muscularization. Previous it has been showed that these changes occur already early during development.
Pericytes are prime candidates to underlie the muscularization of the vasculature in the PH associated with CDH. During growth of the pulmonary vasculature pericytes are recruited in a PDGFβ dependent manner by endothelial cells to stabilize newly formed tubules. As a consequence of the interaction between pericytes and endothelial cells, the pericytes start to release Collagen IV (COLIV), a key component of the basement membrane (BM).
To study the role of pericytes in the development of PH a mouse CDH model was established. First, NG2 was identified as the proper marker for pericytes during lung development. An increase in pericyte coverage was observed with Fluorescence- activated cells sorting and confocal microscopy analysis of immunofluorescent labeled whole mount lung samples from E15 onwards in the CDH mouse model. Reduced expression of COLIV was observed, indicating that the basal membrane between pericytes and endothelial cells is affected in CDH. Furthermore, pericytes in CDH intent to express more smooth muscle actin (ACTA2) and loose proliferation and migration capacity. Samples of CDH patients confirmed that an increase in PDGFRβ expression, and thus an increase in pericytes, is linked to aberrant expression of COLIV at young gestational ages. Thus, the increased pericyte coverage is an important factor in the pathogenesis of CDH and a lack of COLIV in the BM leads to reduced stabilization of the vessel bed and therefore insufficient development of the pulmonary vasculature.
For further understanding of the pathogenesis of PH associated with CDH whole transcriptome analysis on FACS sorted cells was performed at E13 in the CDH mouse model. The transcriptome of four different populations, the epithelial, the mesenchymal, the endothelial and pericytes were analyzed. The analysis of the RNA seq revealed high 158 SHORT TITLE?.
overlap in the differentially expressed genes between the endothelial cell population and the pericyte, indicating that the signaling between endothelial cells and pericytes is affected causing the aberrant vascular development in CDH. Detailed analysis of the RNA sequence data revealed that the expression of the gene Kruppel like factor 4 (Klf4) is reduced in the endothelial cell population in CDH. The reduced expression of KLF4 was confirmed with whole mount fluorescent analysis. KLF4 acts upstream of NOTCH and therefore members of the NOTCH signaling pathway were further analyzed. This confirmed disturbed NOTCH signaling indicating that the aberrant vascular development in CDH is a result of decreased KLF4 expression resulting in disturbed NOTCH signaling.
To explore the new possibilities to treat PH associated with CDH sildenafil was administered to pregnant female rats after they received nitrofen at E9.5, in the canalicular phase of lung development. The administration of sildenafil resulted in improved body weight and improved lung to kidney ratio. Furthermore, the alveolar airspaces increased in diameter, which could be related to the formation of the primary and secondary septa later in prenatal lung development. Moreover, sildenafil reduced the thickening of the SMC layer in arterioles normally present in CDH and prevented the frequently observed aberrant differentiation of pericytes in CDH as indicated by the loss of the colocalization of ACTA2 and PDGFRβ in the capillaries. However, the control group also showed decreases of the alveolar airspaces, Indicating the presence of unwanted side effects in the control group.
A targeted approach for therapy development for CDH should use the knowledge of developmental processes and mechanisms. This thesis describes new detailed insights into the development of the pulmonary vasculature in CDH, revealing increased pericyte coverage, which is the first sign of developing pulmonary hypertension. We furthermore show here that during early lung development (E13), the endothelial cell population in CDH shows the biggest differences in gene expression compared to the other sequenced populations. Down regulation of KLF4 in CDH may explain the reduced extension of the capillary network in CDH.
This work is a first step in revealing the early molecular and cellular mechanisms, which underlie pulmonary hypertension in CDH.

, , ,
D. Tibboel (Dick) , R.J. Rottier (Robbert)
Erasmus University Rotterdam
Cell Biology & Genetics

Kool, H. (2018, January 17). The Cellular Origin of Congenital Diaphragmatic Hernia and Potential Translational Approaches. Retrieved from