Per Lindahl

PDGF-A Signaling Is a Critical Event in Lung Alveolar Myofibroblast Development and Alveogenesis

A mouse platelet-derived growth factor A chain (PDGF-A) null allele is shown to be homozygous lethal, with two distinct restriction points, one prenatally before E10 and one postnatally. Postnatally surviving PDGF-A-deficient mice develop lung emphysema secondary to the failure of alveolar septation. This is apparently caused by the loss of alveolar myofibroblasts and associated elastin fiber deposits. PDGF alpha receptor-positive cells in the lung having the location of putative alveolar myofibroblast progenitors were specifically absent in PDGF-A null mutants. We conclude that PDGF-A is crucial for alveolar myofibroblast ontogeny. We have previously shown that PDGF-B is required in the ontogeny of kidney mesangial cells. The PDGFs therefore appear to regulate the generation of specific populations of myofibroblasts during mammalian development. The two PDGF null phenotypes also reveal analogous morphogenetic functions for myofibroblast-type cells in lung and kidney organogenesis.

Antioxidants Accelerate Lung Cancer Progression in Mice

The antioxidants acetylcysteine and vitamin E accelerate tumor progression and reduce survival in mouse models of lung cancer by disrupting the ROS-p53 axis. The Dark Side of Antioxidants Antioxidants, or chemical compounds that prevent oxidation of other molecules, are widely marketed as dietary supplements with a variety of health claims. One particular characteristic that was frequently attributed to antioxidants is an ability to decrease the risk of cancer. However, a number of studies have shed doubt on this claim in recent years, as emerging evidence has suggested that antioxidants may actually increase the risk of some forms of cancer. Now, a study by Sayin and coauthors sheds new light on this issue and suggests that antioxidants may have a particularly detrimental effect in lung cancer development. When mice carrying mutations that increase their risk of lung cancer were treated with antioxidants, their early precancerous lesions progressed more quickly, and the mice developed more tumors and at more advanced stages. The antioxidants did reduce oxidative stress and DNA damage as expected, but at the same time, they also reduced the expression of p53, a key tumor suppressor protein. This work was done in cells and in mice, but the authors took care to make it as relevant to humans as possible. Thus, the mice were treated with types and doses of antioxidants (vitamin E and acetylcysteine) that healthy humans use, and the results were confirmed in human lung cancer cell lines. Although the current study does not show what would happen to wild-type mice or healthy people using antioxidants, it provides evidence for a procarcinogenic role of antioxidants in people who are already at a higher risk of cancer, such as smokers. Antioxidants are widely used to protect cells from damage induced by reactive oxygen species (ROS). The concept that antioxidants can help fight cancer is deeply rooted in the general population, promoted by the food supplement industry, and supported by some scientific studies. However, clinical trials have reported inconsistent results. We show that supplementing the diet with the antioxidants N-acetylcysteine (NAC) and vitamin E markedly increases tumor progression and reduces survival in mouse models of B-RAF– and K-RAS–induced lung cancer. RNA sequencing revealed that NAC and vitamin E, which are structurally unrelated, produce highly coordinated changes in tumor transcriptome profiles, dominated by reduced expression of endogenous antioxidant genes. NAC and vitamin E increase tumor cell proliferation by reducing ROS, DNA damage, and p53 expression in mouse and human lung tumor cells. Inactivation of p53 increases tumor growth to a similar degree as antioxidants and abolishes the antioxidant effect. Thus, antioxidants accelerate tumor growth by disrupting the ROS-p53 axis. Because somatic mutations in p53 occur late in tumor progression, antioxidants may accelerate the growth of early tumors or precancerous lesions in high-risk populations such as smokers and patients with chronic obstructive pulmonary disease who receive NAC to relieve mucus production.

Vascular endothelial growth factor B controls endothelial fatty acid uptake

The vascular endothelial growth factors (VEGFs) are major angiogenic regulators and are involved in several aspects of endothelial cell physiology. However, the detailed role of VEGF-B in blood vessel function has remained unclear. Here we show that VEGF-B has an unexpected role in endothelial targeting of lipids to peripheral tissues. Dietary lipids present in circulation have to be transported through the vascular endothelium to be metabolized by tissue cells, a mechanism that is poorly understood. Bioinformatic analysis showed that Vegfb was tightly co-expressed with nuclear-encoded mitochondrial genes across a large variety of physiological conditions in mice, pointing to a role for VEGF-B in metabolism. VEGF-B specifically controlled endothelial uptake of fatty acids via transcriptional regulation of vascular fatty acid transport proteins. As a consequence, Vegfb-/- mice showed less uptake and accumulation of lipids in muscle, heart and brown adipose tissue, and instead shunted lipids to white adipose tissue. This regulation was mediated by VEGF receptor 1 and neuropilin 1 expressed by the endothelium. The co-expression of VEGF-B and mitochondrial proteins introduces a novel regulatory mechanism, whereby endothelial lipid uptake and mitochondrial lipid use are tightly coordinated. The involvement of VEGF-B in lipid uptake may open up the possibility for novel strategies to modulate pathological lipid accumulation in diabetes, obesity and cardiovascular diseases.

The effect of rotenone on respiration and its point of attack

The action of rotenone on the respiration of fish gill filaments from Leuciscus rutilus (L.), mouse liver slices, and rat liver mitochondria has been studied. 1. 1. Exposure of intact and excised gill filaments to rotenone induces an inhibition of their oxygen consumption. This effect was more pronounced when the gill filaments were brought into contact with the rotenone in vivo. 2. 2. Rotenone also induced a decrease of the oxygen uptake of mouse liver slices, but in order to obtain the same degree of inhibition in liver slices as in gill filaments, the concentration of rotenone had to be increased 50 times. 3. 3. In isolated rat liver mitochondria the aerobic oxidation of pyruvate was almost completely inhibited by 6.0 × 10−7 M rotenone, but in the same experiment this concentration had no effect on the oxidation of succinate. The inhibition of the oxidation of pyruvate induced by rotenone was decreased to 14 per cent by addition of methylene blue. 4. 4. Addition of methylene blue reduced the inhibition of the oxygen uptake of gill filaments caused by rotenone after excision, as well as in vivo. 5. 5. Uncoupling of the phosphorylation by 10−4 M DNP did not release the inhibition of mitochondrial respiration caused by rotenone. 6. 6. By investigating the difference spectra of the components of the respiratory chain in a mitochondrial suspension it was found that the diaphorase, cytochromes b, c, a, and a3 were oxidized in the presence of rotenone. 7. 7. These results indicate that rotenone blocks in the electron transporting system a link which is situated at the diaphorase level.

Transcription Profiling of Platelet-Derived Growth Factor-B-Deficient Mouse Embryos Identifies RGS5 as a Novel Marker for Pericytes and Vascular Smooth Muscle Cells

All blood capillaries consist of endothelial tubes surrounded by mural cells referred to as pericytes. The origin, recruitment, and function of the pericytes is poorly understood, but the importance of these cells is underscored by the severe cardiovascular defects in mice genetically devoid of factors regulating pericyte recruitment to embryonic vessels, and by the association between pericyte loss and microangiopathy in diabetes mellitus. A general problem in the study of pericytes is the shortage of markers for these cells. To identify new markers for pericytes, we have taken advantage of the platelet-derived growth factor (PDGF)-B knockout mouse model, in which developing blood vessels in the central nervous system are almost completely devoid of pericytes. Using cDNA microarrays, we analyzed the gene expression in PDGF-B null embryos in comparison with corresponding wild-type embryos and searched for down-regulated genes. The most down-regulated gene present on our microarray was RGS5, a member of the RGS family of GTPase-activating proteins for G proteins. In situ hybridization identified RGS5 expression in brain pericytes, and in pericytes and vascular smooth muscle cells in certain other, but not all, locations. Absence of RGS5 expression in PDGF-B and PDGFR beta-null embryos correlated with pericyte loss in these mice. Residual RGS5 expression in rare pericytes suggested that RGS5 is a pericyte marker expressed independently of PDGF-B/R beta signaling. With RGS5 as a proof-of-principle, our data demonstrate the usefulness of microarray analysis of mouse models for abnormal pericyte development in the identification of new pericyte-specific markers.

Antioxidants can increase melanoma metastasis in mice

Antioxidants increase migration and invasion of human melanoma cells and accelerate metastasis in an endogenous mouse model of malignant melanoma. Another strike against antioxidants Antioxidants are found in a variety of foods and dietary supplements and are frequently used with the goal of preventing cancer, but mounting evidence suggests that they may not be as beneficial as once thought. Clinical studies have shown mixed or no benefits, and other works demonstrated that antioxidants may accelerate the progression of lung cancer. Now, Le Gal et al. discovered that some common antioxidants increase the rate of melanoma cell migration and invasion and increase metastasis in a mouse model. These are early findings, and additional work will be required to confirm the generalizability of this observation. Nevertheless, the results suggest a need for caution in the use of antioxidants, especially for patients with existing cancer. Antioxidants in the diet and supplements are widely used to protect against cancer, but clinical trials with antioxidants do not support this concept. Some trials show that antioxidants actually increase cancer risk and a study in mice showed that antioxidants accelerate the progression of primary lung tumors. However, little is known about the impact of antioxidant supplementation on the progression of other types of cancer, including malignant melanoma. We show that administration of N-acetylcysteine (NAC) increases lymph node metastases in an endogenous mouse model of malignant melanoma but has no impact on the number and size of primary tumors. Similarly, NAC and the soluble vitamin E analog Trolox markedly increased the migration and invasive properties of human malignant melanoma cells but did not affect their proliferation. Both antioxidants increased the ratio between reduced and oxidized glutathione in melanoma cells and in lymph node metastases, and the increased migration depended on new glutathione synthesis. Furthermore, both NAC and Trolox increased the activation of the small guanosine triphosphatase (GTPase) RHOA, and blocking downstream RHOA signaling abolished antioxidant-induced migration. These results demonstrate that antioxidants and the glutathione system play a previously unappreciated role in malignant melanoma progression.

Developmental origin of smooth muscle cells in the descending aorta in mice

Aortic smooth muscle cells (SMCs) have been proposed to derive from lateral plate mesoderm. It has further been suggested that induction of SMC differentiation is confined to the ventral side of the aorta, and that SMCs later migrate to the dorsal side. In this study, we investigate the origin of SMCs in the descending aorta using recombination-based lineage tracing in mice. Hoxb6-cre transgenic mice were crossed with Rosa 26 reporter mice to track cells of lateral plate mesoderm origin. The contribution of lateral plate mesoderm to SMCs in the descending aorta was determined at different stages of development. SMC differentiation was induced in lateral plate mesoderm-derived cells on the ventral side of the aorta at embryonic day (E) 9.0-9.5, as indicated by expression of the SMC-specific reporter gene SM22α-lacZ. There was, however, no migration of SMCs from the ventral to the dorsal side of the vessel. Moreover, the lateral plate mesoderm-derived cells in the ventral wall of the aorta were replaced by somite-derived cells at E10.5, as indicated by reporter gene expression in Meox1-cre/Rosa 26 double transgenic mice. Examination of reporter gene expression in adult aortas from Hoxb6-cre/Rosa 26 and Meox1-cre/Rosa 26 double transgenic mice suggested that all SMCs in the adult descending aorta derive from the somites, whereas no contribution was recorded from lateral plate mesoderm.

ENDOTHELIAL-PERIVASCULAR CELL SIGNALING IN VASCULAR DEVELOPMENT : LESSONS FROM KNOCKOUT MICE

The importance of perivascular-endothelial cell interactions during blood vessel development is discussed in the light of recent findings in platelet-derived growth factor-B, platelet-derived growth factor receptor-beta, angiopoietin-1 and tie-2 knockout mice, which all show deficient development of perivascular cells. The initial formation of networks of endothelial tubes (vasculogenesis) does not seem to depend on the perivascular cells but subsequent vessel remodeling relies on mesenchymal-endothelial short-range signaling. Based on findings in platelet-derived growth factor and platelet-derived growth factor receptor knockout mice, a general model for the role of platelet-derived growth factors in smooth muscle development is proposed.

Microarray analysis of blood microvessels from PDGF-B and PDGF-Rβ mutant mice identifies novel markers for brain pericytes

Normal blood microvessels are lined by pericytes, which contribute to microvessel development and stability through mechanisms that are poorly understood. Pericyte deficiency has been implicated in the pathogenesis of microvascular abnormalities associated with diabetes and tumors. However, the unambiguous identification of pericytes is still a problem because of cellular heterogeneity and few available molecular markers. Here we describe an approach to identify pericyte markers based on transcription profiling of pericytedeficient brain microvessels isolated from platelet‐derived growth factor (PDGF‐B) –/– and PDGF beta receptor (PDGFRβ) –/– mouse mutants. The approach was validated by the identification of known pericyte markers among the most down‐regulated genes in PDGF‐B –/– and PDGFRβ –/– microvessels. Of candidates for novel pericyte markers, we selected ATP‐sensitive potassiumchannel Kir6.1 (also known as Kcnj8) and sulfonylurea receptor 2, (SUR2, also known as Abcc9), both part of the same channel complex, as well as delta homologue 1 (DLK1) for in situ hybridization, which demonstrated their specific expression in brain pericytes of mouse embryos. We also show that Kir6.1 is highly expressed in pericytes in brain but undetectable in pericytes in skin and heart. The three new brain pericyte markers are signaling molecules implicated in ion transport and intercellular signaling, potentially opening new windows on pericyte function in brain microvessels.—Bondjers, C., He, L., Takemoto, M., Norlin, J., Asker, N., Hellström, M., Lindahl, P., Betsholtz, C. Microarray analysis of blood microvessels from PDGF‐B and PDGF‐Rβ mutant mice identifies novel markers for brain pericytes. FASEB J. 20, E1005–1013 (2006)

mRNA Expression Profiling of Laser Microbeam Microdissected Cells from Slender Embryonic Structures

Microarray hybridization has rapidly evolved as an important tool for genomic studies and studies of gene regulation at the transcriptome level. Expression profiles from homogenous samples such as yeast and mammalian cell cultures are currently extending our understanding of biology, whereas analyses of multicellular organisms are more difficult because of tissue complexity. The combination of laser microdissection, RNA amplification, and microarray hybridization has the potential to provide expression profiles from selected populations of cells in vivo. In this article, we present and evaluate an experimental procedure for global gene expression analysis of slender embryonic structures using laser microbeam microdissection and laser pressure catapulting. As a proof of principle, expression profiles from 1000 cells in the mouse embryonic (E9.5) dorsal aorta were generated and compared with profiles for captured mesenchymal cells located one cell diameter further away from the aortic lumen. A number of genes were overexpressed in the aorta, including 11 previously known markers for blood vessels. Among the blood vessel markers were endoglin, tie-2, PDGFB, and integrin-beta1, that are important regulators of blood vessel formation. This demonstrates that microarray analysis of laser microbeam micro-dissected cells is sufficiently sensitive for identifying genes with regulative functions.