8 research outputs found
Identification of suitable reference genes for normalization of reverse transcription quantitative real-time PCR (RT-qPCR) in the fibrotic phase of the bleomycin mouse model of pulmonary fibrosis
Abstract
Idiopathic pulmonary fibrosis (IPF) is a severe lung disease with a poor prognosis and few treatment options. In the most widely used experimental model for this disease, bleomycin is administered into the lungs of mice, causing a reaction of inflammation and consequent fibrosis that resembles the progression of human IPF. The inflammation and fibrosis together induce changes in gene expression that can be analyzed with reverse transcription quantitative real-time PCR (RT-qPCR), in which accurate normalization with a set of stably expressed reference genes is critical for obtaining reliable results. This work compares ten commonly used candidate reference genes in the late, fibrotic phase of bleomycin-induced pulmonary fibrosis and ranks them from the most to the least stable using NormFinder and geNorm. Sdha, Polr2a and Hprt were identified as the best performing and least variable reference genes when alternating between normal and fibrotic conditions. In order to validate the findings, we investigated the expression of Tnf and Col1a1, representing the hallmarks of inflammation and fibrotic changes, respectively. With the best three genes as references, both were found to be upregulated relative to untreated controls, unlike the situation when analyzed solely with Gapdh, a commonly used reference gene. We therefore recommend Sdha, Polr2a and Hprt as reference genes for RT-qPCR in the 4-week bleomycin challenge that represents the late fibrotic phase
Expression and roles of individual HIF prolyl 4-hydroxylase isoenzymes in the regulation of the hypoxia response pathway along the murine gastrointestinal epithelium
Abstract
The HIF prolyl 4-hydroxylases (HIF-P4H) control hypoxia-inducible factor (HIF), a powerful mechanism regulating cellular adaptation to decreased oxygenation. The gastrointestinal epithelium subsists in âphysiological hypoxiaâ and should therefore have an especially well-designed control over this adaptation. Thus, we assessed the absolute mRNA expression levels of the HIF pathway components, Hif1a, HIF2a, Hif-p4h-1, 2 and 3 and factor inhibiting HIF (Fih1) in murine jejunum, caecum and colon epithelium using droplet digital PCR. We found a higher expression of all these genes towards the distal end of the gastrointestinal tract. We detected mRNA for Hif-p4h-1, 2 and 3 in all parts of the gastrointestinal tract. Hif-p4h-2 had significantly higher expression levels compared to Hif-p4h-1 and 3 in colon and caecum epithelium. To test the roles each HIF-P4H isoform plays in the gut epithelium, we measured the gene expression of classical HIF target genes in Hif-p4h-1â/â, Hif-p4h-2 hypomorph and Hif-p4h-3â/â mice. Only Hif-p4h-2 hypomorphism led to an upregulation of HIF target genes, confirming a predominant role of HIF-P4H-2. However, the abundance of Hif-p4h-1 and 3 expression in the gastrointestinal epithelium implies that these isoforms may have specific functions as well. Thus, the development of selective inhibitors might be useful for diverging therapeutic needs
Deletion of hypoxia-inducible factor prolyl 4-hydroxylase 2 in FoxD1-lineage mesenchymal cells leads to congenital truncal alopecia
Abstract
Hypoxia-inducible factors (HIFs) induce numerous genes regulating oxygen homeostasis. As oxygen sensors of the cells, the HIF prolyl 4-hydroxylases (HIF-P4Hs) regulate the stability of HIFs in an oxygen-dependent manner. During hair follicle (HF) morphogenesis and cycling, the location of dermal papilla (DP) alternates between the dermis and hypodermis and results in varying oxygen levels for the DP cells. These cells are known to express hypoxia-inducible genes, but the role of the hypoxia response pathway in HF development and homeostasis has not been studied. Using conditional gene targeting and analysis of hair morphogenesis, we show here that lack of Hif-p4h-2 in Forkhead box D1 (FoxD1)-lineage mesodermal cells interferes with the normal HF development in mice. FoxD1-lineage cells were found to be mainly mesenchymal cells located in the dermis of truncal skin, including those cells composing the DP of HFs. We found that upon Hif-p4h-2 inactivation, HF development was disturbed during the first catagen leading to formation of epithelial-lined HF cysts filled by unorganized keratins, which eventually manifested as truncal alopecia. Furthermore, the depletion of Hif-p4h-2 led to HIF stabilization and dysregulation of multiple genes involved in keratin formation, HF differentiation, and HIF, transforming growth factor ÎČ (TGF-ÎČ), and Notch signaling. We hypothesize that the failure of HF cycling is likely to be mechanistically caused by disruption of the interplay of the HIF, TGF-ÎČ, and Notch pathways. In summary, we show here for the first time that HIF-P4H-2 function in FoxD1-lineage cells is essential for the normal development and homeostasis of HFs
Contribution of HIF-P4H isoenzyme inhibition to metabolism indicates major beneficial effects being conveyed by HIF-P4H-2 antagonism
Abstract
Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases (HIF-P4Hs 1â3) are druggable targets in renal anemia, where pan-HIF-P4H inhibitors induce an erythropoietic response. Preclinical data suggest that HIF-P4Hs could also be therapeutic targets for treating metabolic dysfunction, although the contributions of HIF-P4H isoenzymes in various tissues to the metabolic phenotype are inadequately understood. Here, we used mouse lines that were gene-deficient for HIF-P4Hs 1 to 3 and two preclinical pan-HIF-P4H inhibitors to study the contributions of these isoenzymes to the anthropometric and metabolic outcome and HIF response. We show both inhibitors induced a HIF response in wildtype white adipose tissue (WAT), liver, and skeletal muscle and alleviated metabolic dysfunction during a 6-week treatment period, but they did not alter healthy metabolism. Our data indicate that HIF-P4H-1 contributed especially to skeletal muscle and WAT metabolism and that its loss lowered body weight and serum cholesterol levels upon aging. In addition, we found HIF-P4H-3 had effects on the liver and WAT and its loss increased body weight, adiposity, liver weight and triglyceride levels, WAT inflammation, and cholesterol levels and resulted in hyperglycemia and insulin resistance, especially during aging. Finally, we demonstrate HIF-P4H-2 affected all tissues studied; its inhibition lowered body and liver weight and serum cholesterol levels and improved glucose tolerance. We found very few HIF target metabolic mRNAs were regulated by the inhibition of three isoenzymes, thus suggesting a potential for selective therapeutic tractability. Altogether, these data provide specifications for the future development of HIF-P4H inhibitors for the treatment of metabolic diseases
PHD2 deletion in endothelial or arterial smooth muscle cells reveals vascular cell type-specific responses in pulmonary hypertension and fibrosis
Abstract
Hypoxia plays an important regulatory role in the vasculature to adjust blood flow to meet metabolic requirements. At the level of gene transcription, the responses are mediated by hypoxia-inducible factor (HIF) the stability of which is controlled by the HIF prolyl 4-hydroxylase-2 (PHD2). In the lungs hypoxia results in vasoconstriction, however, the pathophysiological relevance of PHD2 in the major arterial cell types; endothelial cells (ECs) and arterial smooth muscle cells (aSMCs) in the adult vasculature is incompletely characterized. Here, we investigated PHD2-dependent vascular homeostasis utilizing inducible deletions of PHD2 either in ECs (Phd2âECi) or in aSMCs (Phd2âaSMC). Cardiovascular function and lung pathologies were studied using echocardiography, Doppler ultrasonography, intraventricular pressure measurement, histological, ultrastructural, and transcriptional methods. Cell intrinsic responses were investigated in hypoxia and in conditions mimicking hypertension-induced hemodynamic stress. Phd2âECi resulted in progressive pulmonary disease characterized by a thickened respiratory basement membrane (BM), alveolar fibrosis, increased pulmonary artery pressure, and adaptive hypertrophy of the right ventricle (RV). A low oxygen environment resulted in alterations in cultured ECs similar to those in Phd2âECi mice, involving BM components and vascular tone regulators favoring the contraction of SMCs. In contrast, Phd2âaSMC resulted in elevated RV pressure without alterations in vascular tone regulators. Mechanistically, PHD2 inhibition in aSMCs involved actin polymerization -related tension development via activated cofilin. The results also indicated that hemodynamic stress, rather than PHD2-dependent hypoxia response alone, potentiates structural remodeling of the extracellular matrix in the pulmonary microvasculature and respiratory failure
Blocking surgically induced lysyl oxidase activity reduces the risk of lung metastases
Abstract
Surgery remains the most successful curative treatment for cancer. However, some patients with early-stage disease who undergo surgery eventually succumb to distantmetastasis. Here, we show that in response to surgery, the lungs become more vulnerable to metastasis due to extracellular matrix remodeling. Mice that undergo surgery or that are preconditioned with plasma from donor mice that underwent surgery succumb to lung metastases earlier than controls. Increased lysyl oxidase (LOX) activity and expression, fibrillary collagen crosslinking, and focal adhesion signaling contribute to this effect, with the hypoxic surgical site serving as the source of LOX. Furthermore, the lungs of recipient mice injected with plasma from post-surgical colorectal cancer patients are more prone to metastatic seeding than mice injected with baseline plasma. Downregulation of LOX activity or levels reduces lung metastasis after surgery and increases survival, highlighting the potential of LOX inhibition in reducing the risk of metastasis following surgery
Upregulated integrin α11 in the stroma of cutaneous squamous cell carcinoma promotes skin carcinogenesis
Abstract
Integrin α11ÎČ1 is a collagen-binding integrin that is needed to induce and maintain the myofibroblast phenotype in fibrotic tissues and during wound healing. The expression of the α11 is upregulated in cancer-associated fibroblasts (CAFs) in various human neoplasms. We investigated α11 expression in human cutaneous squamous cell carcinoma (cSCC) and in benign and premalignant human skin lesions and monitored its effects on cSCC development by subjecting α11-knockout (Itga11â/â) mice to the DMBA/TPA skin carcinogenesis protocol. α11-deficient mice showed significantly decreased tumor cell proliferation, leading to delayed tumor development and reduced tumor burden. Integrin α11 expression was significantly upregulated in the desmoplastic tumor stroma of human and mouse cSCCs, and the highest α11 expression was detected in high-grade tumors. Our results point to a reduced ability of α11-deficient stromal cells to differentiate into matrix-producing and tumor-promoting CAFs and suggest that this is one causative mechanism underlying the observed decreased tumor growth. An unexpected finding in our study was that, despite reduced CAF activation, the α11-deficient skin tumors were characterized by the presence of thick and regularly aligned collagen bundles. This finding was attributed to a higher expression of TGFÎČ1 and collagen crosslinking lysyl oxidases in the Itga11â/â tumor stroma. In summary, our data suggest that α11ÎČ1 operates in a complex interactive tumor environment to regulate ECM synthesis and collagen organization and thus foster cSCC growth. Further studies with advanced experimental models are still needed to define the exact roles and molecular mechanisms of stromal α11ÎČ1 in skin tumorigenesis
Lysyl Oxidase Oxidizes Cell Membrane Proteins and Enhances the Chemotactic Response of Vascular Smooth Muscle Cells*
Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied
cell types. Here we demonstrate that inhibition of LOX activity by
ÎČ-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells
(SMCs) reduced the chemotactic response and sensitivity of these cells toward
LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly
enhanced in the presence of a non-chemotactic concentration of LOX. We
considered the possibility that extracellular LOX may oxidize cell surface
proteins, including the PDGF receptor-ÎČ (PDGFR-ÎČ), to affect
PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC
contained oxidized PDGFR-ÎČ. The oxidation of this receptor and other
membrane proteins was largely prevented in cells preincubated with BAPN.
Addition of purified LOX to these cells restored the profile of oxidized
proteins toward that of control cells. The high affinity and capacity for the
binding of PDGF-BB by cells containing oxidized PDGFR-ÎČ was diminished by
âŒ2-fold when compared with cells in which oxidation by LOX was prevented
by BAPN. Phosphorylated members of the PDGFR-ÎČ-dependent signal
transduction pathway, including PDGFR-ÎČ, SHP2, AKT1, and ERK1/ERK2
(p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX
knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs
treated with BAPN. These novel findings suggest that LOX activity is essential
to generate optimal chemotactic sensitivity of cells to chemoattractants by
oxidizing specific cell surface proteins, such as PDGFR-ÎČ