Deterioration of alveolar development in mice with both HIF-3α knockout and HIF-2α knockdown

Objective Earlier studies from our group using hypoxia-inducible factor 3α knockout mice showed impairments in lung remodeling and lung endothelial cells. Another research from our group demonstrated that impaired expression of hypoxia-inducible factor 2α induced compensatory expression of hypoxia-inducible factor 1α in hypoxia-inducible factor 2α knockdown mice. The present study uncovers more insights by extending the investigation, utilizing mice with both hypoxia-inducible factor 3α knockout and hypoxia-inducible factor 2α knockdown. Results No mice with both hypoxia-inducible factor 3α knockout and hypoxia-inducible factor 2α knockdown died immediately after birth. The mice with both hypoxia-inducible factor 3α knockout and hypoxia-inducible factor 2α knockdown exhibited impaired alveolar sacs and lung alveolar structure and decreased endothelial cell numbers. Analysis of relative mRNA expression revealed depressed expressions of hypoxia-inducible factor 1α, vascular cell adhesion molecule 1, vascular endothelial cadherin, angiopoietin 2, Tie-2, and vascular endothelial growth factor in the lungs of mice with both hypoxia-inducible factor 3α knockout and hypoxia-inducible factor 2α knockdown compared to that in wild-type mice. Further analysis is needed to elucidate the impaired development occurred in the lung endothelial cells.


Introduction
Hypoxia-inducible factors (HIFs) are heterodimers that consist of three oxygen-sensitive α-subunits (HIF-1α, HIF-2α, and HIF-3α) and a β-subunit, the aryl hydrocarbon receptor nuclear translocator (ARNT). HIFs act as regulators of the molecular hypoxic response [1,2]; in a study examining normal alveolarization in fostered newborn rats, HIFs promoted alveolar development and regeneration by preventing and repairing oxygeninduced alveolar damage [3]. Nonetheless, HIF-1α inhibition using antisense knockdown in vitro during early lung development decreased vascular development and epithelial branching morphogenesis in lung explants [4]. In contrast, the conditional overexpression of HIF-1α in embryonic lung epithelium also impaired branching morphogenesis and lung maturation and affected vascular lung abnormalities, including hemorrhages and increased lymphangiogenesis [5]. Collectively, these data suggest that interference in the alveolar epithelium by oxygen pressure changes, including hypoxia, can affect alveolar homeostasis, leading to epithelial injuries and diseases such as lung fibrosis [6][7][8][9]. A previous study from our group that used HIF-3α knockout (−/−) mice showed impaired lung remodeling exhibited by the walls of the secondary septa in subdivided alveoli, and immunostaining of alveolar endothelial cells presented an increase in defective space in the interalveolar septa and hyperplasia of endothelial cells during the maturation of alveolar formation in these knockout mice [10]. Additionally, another study from our group revealed that these HIF-3α −/− mice showed impairments in lung endothelial cells presented by slow growth and a decreased number of tubes formed by endothelial cells [11]. Furthermore, a  [12]. The present study uncovers more insights by extending the investigation from those previously stated findings of our group utilizing HIF-3α −/− and HIF-2α kd/kd (double-mutant) mice. In this study, male and female HIF-3α −/− and HIF-2α knockdown heterozygote (kd/+) mice were interbred, resulting in the doublemutant mice previously mentioned.

Materials and methods Mice
All of the experiments performed were approved by the ethics committee of the University of Tsukuba. All wildtype (WT) and mutant mouse lines were of the C57BL/6J genetic background. HIF-2α kd/kd mice were generated as previously reported [13]. HIF-3α −/− mice were obtained as previously published [11]. Mating of 12 pairs of HIF-3α −/− and HIF-2α kd/+ mice for a breeding period of 1 year generated the double-mutant mice. The genome DNA was extract from tail of neonatal pup. The genotype of mouse was determined by polymerase chain reaction (PCR) as described previously [11,13].

Quantitative reverse transcription polymerase chain reaction (qPCR)
Total RNA was obtained from samples (n = 3) with the use of the extraction reagent (Sepasol-RNA I Super G; Nakalai Tesque, Kyoto, Japan). cDNA was then synthesized by reverse transcription (ReveTra Ace; TOYOBO, Osaka, Japan). The expression level was analyzed by using the 7500 Fast Real-Time PCR machine (Applied Biosystems, Carlsbad, CA, USA) with SYBR-green (Life Technologies, Carlsbad, CA, USA). Experiments were performed in triplicate, and the resulting data were analyzed by the delta CT method.

The double-mutant mice had impaired alveolar sacs and lung alveolar structure and decreased endothelial cell numbers
Hematoxylin and eosin staining of the lung in WT, HIF-3α −/−, and double-mutant mice was performed at P0 (Fig. 1). We found that alveolar sacs were almost imperceptible in the lungs of double-mutant mice, and the appearance of the blood vessels in the lungs of these mice was different than that in the lungs of HIF-3α −/− and WT mice. There is no phenotype data for HIF-2α kd/kd mice at present. Moreover, immunohistochemistry analysis of sections from neonatal WT and doublemutant mice at 2-3 days of age (Fig. 2) by CD31 staining showed impaired lung vessel structure of the neonatal double-mutant mice accompanied with the decreased of endothelial cell numbers. HIF-1α and HIF-2α staining of the neonatal double-mutant mice revealed the decreased expressions of both HIFs and endothelial cell numbers. VCAM-1 and VE-cadherin staining of the neonatal double-mutant mice showed their expressions and also the decreased of endothelial cell numbers.

Discussion
The present study has elucidated that no double-mutant mice died immediately after birth. This new finding is slightly opposed to the common knowledge that HIF deficiency is immediately postnatally lethal. The reason that none of the double-mutant mice died postnatally remains unclear. Furthermore, this study is the first report that the alveolar sacs of the double-mutant mice are impaired. The current result has broadened the understanding from our previous study that revealed incomplete alveolar spaces in HIF-3α −/− mice [10]. Immunohistochemistry results have shown that the decreased of endothelial cell numbers, which impair proliferative and angiogenic activities appeared to contribute to impaired lung alveolar structure of the neonatal double-mutant mice. In addition, the lung endothelial cells isolated from the neonatal double-mutant mice showed the impaired proliferative ability (data not shown), suggesting the functional impairment of these cells by both HIF-3α −/− and HIF-2α kd/kd. The decreased of HIF-1α and HIF-2α expressions causing of lacking to overcome the happening of oxygen homeostasis disruption also deteriorate such lung alveolar structure. Such pathological features may provide further insight into the molecular mechanism of alveolar development especially  Fig. 2 The double-mutant mice had impaired lung alveolar structure and decreased endothelial cell numbers. Immunohistochemistry analysis performed to sections from neonatal WT and double-mutant mice at 2-3 days of age using CD31, HIF-1α, HIF-2α, VE-cadherin, and VCAM-1 staining. HIF-1α, hypoxia-inducible factor 1α; HIF-2α, hypoxia-inducible factor 2α; H3, hypoxia-inducible factor 3α; H2, hypoxia-inducible factor 2α; ::, intercrossed with; −/−, knockout; kd/kd, knockdown; VE-cadherin, vascular endothelial cadherin; VCAM-1, vascular cell adhesion molecule 1; WT, wild-type for further investigation at the embryonic stage. We next acknowledged that our current results on the gene expressions in the lung of the neonatal HIF-3α −/− mice are partly in line as well as in contrary to the results showed on our former study utilizing the adult HIF-3α −/− mice [11]. The contrary results in the current study are mostly correlated to the angiogenic gene regulations due to the depressed mRNA levels of HIF-1α, HIF-2α, VCAM-1, VE-cadherin, Ang-2, Tie-2, HIF-2α, VEGF, and Flk-1 in the lung of the neonatal HIF-3α −/− mice. The mRNA levels of HIF-1α, VCAM-1, VE-cadherin, Ang-2, Tie-2, and VEGF are even more depressed in the lung of the neonatal double-mutant mice. Our understanding of these new findings may be further understood in light Fig. 3 The double-mutant mouse lung exhibited depressed expressions of HIF-1α, VCAM-1, VE-cadherin, Ang-2, Tie-2, and VEGF. The utilizing qPCR performed the analysis of mRNA relative expressions for HIF-1α, HIF-2α, VCAM-1, VE-cadherin, Ang-1, Ang-2, Tie-2, VGEF, and Flk-1 that normalized to β-actin. Ang-1, angiopoietin 1; Ang-2, angiopoietin 2; H3, hypoxia-inducible factor 3α; H2, hypoxia-inducible factor 2α; ::, intercrossed with; −/−, knockout; kd/kd, knockdown; HIF-1α, hypoxia-inducible factor 1α; HIF-2α, hypoxia-inducible factor 2α; mRNA, messenger ribonucleic acid; VCAM-1, vascular cell adhesion molecule 1; VE-cadherin, vascular endothelial cadherin; VEGF, vascular endothelial growth factor; WT, wild-type of knowledge that the VEGF/Flk-1 and angiopoietin/ Tie-2 signaling pathways are vital for the maintenance of endothelial cell homeostasis [16][17][18], which then can explain impaired alveolar sacs and lung alveolar structure conditions. Ang-1 that is interestingly highly expressed even though HIF-1α is inversely expressed in the lung of the neonatal double-mutant mice, which against the previous report concluding HIF-1α explicitly targets Ang-1 [19] may indicate other regulations involved. Overall, these findings should be investigated further to firmly elucidate the impaired development occurred in the lung endothelial cells.

Limitations
The limited number of double-mutant mice that survive postnatally may hinder the acquisition of sufficient samples for the isolation and identification of the lung endothelial cells.

Authors' contributions
FZA and TY contributed equally to this work. FZA conceptualized the study; analyzed and interpreted all immunohistochemistry and section staining and qPCR data, and was a major contributor in writing the manuscript. TY designed the study on the lung by using double-mutant mice; performed breeding of all the mice, isolated all lung tissue samples, and performed and analyzed all immunohistochemistry and section staining and qPCR; and critically revised the manuscript. OO designed the study on the lung by using double-mutant mice and critically revised the manuscript. All authors read and approved the final manuscript.