Publications

Urokinase-type plasminogen activator receptor (uPAR, or CD87) is a key component of the fibrinolytic system involved in extracellular matrix remodeling and angiogenesis. The cleavage/inactivation of uPAR is a crucial step in fibroblast-to-myofibroblast transition and has been implicated in systemic sclerosis (SSc, scleroderma) peripheral microvasculopathy and impaired angiogenesis. In the present study, we investigated whether uPAR gene inactivation in mice could result in tissue fibrosis and peripheral microvasculopathy resembling human SSc. Skin and lung sections from uPAR-deficient mice and wild-type littermates at 12 and 24 weeks of age were stained with hematoxylin-eosin, Masson’s trichrome and Picrosirius red. Dermal thickness and hydroxyproline content were quantified. The number of myofibroblasts and microvessels were determined in skin sections immunostained for alpha-smooth muscle actin and the pan-endothelial cell marker CD31, respectively. Endothelial cell apoptosis was assessed by TUNEL/CD31 immunofluorescence assay. Dermal thickness, collagen content and myofibroblast counts were significantly greater in uPAR-deficient mice than in wild-type littermates. In uPAR-deficient mice, dermal fibrosis was paralleled by endothelial cell apoptosis and severe loss of microvessels. Lung specimens from uPAR-deficient mice exhibited non-specific interstitial pneumonia-like pathological features with large patchy areas of lung parenchyma displaying a uniform interstitial involvement characterized by both diffuse cellular inflammation and collagen deposition. In uPAR-deficient mice pulmonary pathology worsened significantly from 12 to 24 weeks of age, as shown by a significant increase in alveolar septal width and collagen content.The absence of uPAR induces dermal and pulmonary fibrosis and peripheral microvasculopathy in mice closely mimicking human SSc. uPAR-deficient mice might be a promising preclinical model to study the pathogenetic mechanisms of human SSc and enable testing of antifibrotic agents and drugs targeting small vessel vasculopathy simultaneously.

Background: Inhibition of angiogenesis is currently hot topic in the search for an effective treatment against hepatocellular carcinoma (HCC). Placental growth factor (PlGF) is a VEGF analogue only involved in pathologic angiogenesis and its inhibition has the potential to restrain tumour growth, without affecting healthy organs. Therefore, we assessed whether administration of PlGF-antibodies could serve as a potential therapy for HCC in mice.\n\nMethods: 5-week-old male mice received intraperitoneal injections with diethylnitrosamine (35 mg/kg bodyweight, 1x/week) leading to HCC after 25W. At 26W mice were treated with murine PlGF-antibodies (20 mg/kg aPlGF, 2x/week) or IgG for 5W. Tumour vasculature was assessed with endoglin staining. As a marker for vessel abnormality the number of cords without lumen, tortuous vessels and intercapillary distances were quantified. Arterialisation was assessed (aSMA staining) and the number and size of unpaired arteries (i.e. large SMA+ vessels not accompanied by a bile duct) determined. Stainings for HIF2alpha and PCNA were used to identify hypoxia and proliferation. \n\nResults: treatment with aPlGF caused a significant decrease in mortality (45% in IgG vs 23% aPlGF mice; P< 0.05) and lowered (p< 0,05) tumour burden. The vascular network decreased around the PlGF-treated tumours (P< 0,01) ; fewer (p< 0,05) and smaller (p< 0,001) unpaired arteries were present and PlGF-blockage partially normalized the vasculature. These changes were functionally relevant, as PlGF-blocked HCC nodules expressed lower levels of HIF2 (p< 0,05) and showed less proliferation (p< 0,05).\n\nConclusion: Inhibition of PlGF has a positive effect on survival in mice with HCC. aPlGF treatment decreased the tumour induced vascularisation and arterialisation, without effecting healthy tissue. Furthermore, the normalised vasculature and reduced hypoxia, decreases the proliferation of malignant cells. Given that VEGF-inhibitors enhance metastasis by evoking hypoxia, the anti-abnormalization activity of aPlGF may offer promising target to combat HCC.\n\nMethods: 5-week-old male mice received intraperitoneal injections with diethylnitrosamine (35 mg/kg bodyweight, 1x/week) leading to HCC after 25W. At 26W mice were treated with murine PlGF-antibodies (20 mg/kg aPlGF, 2x/week) or IgG for 5W. Tumour vasculature was assessed with endoglin staining. As a marker for vessel abnormality the number of cords without lumen, tortuous vessels and intercapillary distances were quantified. Arterialisation was assessed (aSMA staining) and the number and size of unpaired arteries (i.e. large SMA+ vessels not accompanied by a bile duct) determined. Stainings for HIF2alpha and PCNA were used to identify hypoxia and proliferation. \n\nResults: treatment with aPlGF caused a significant decrease in mortality (45% in IgG vs 23% aPlGF mice; P< 0.05) and lowered (p< 0,05) tumour burden. The vascular network decreased around the PlGF-treated tumours (P< 0,01) ; fewer (p< 0,05) and smaller (p< 0,001) unpaired arteries were present and PlGF-blockage partially normalized the vasculature. These changes were functionally relevant, as PlGF-blocked HCC nodules expressed lower levels of HIF2 (p< 0,05) and showed less proliferation (p< 0,05).\n\nConclusion: Inhibition of PlGF has a positive effect on survival in mice with HCC. aPlGF treatment decreased the tumour induced vascularisation and arterialisation, without effecting healthy tissue. Furthermore, the normalised vasculature and reduced hypoxia, decreases the proliferation of malignant cells. Given that VEGF-inhibitors enhance metastasis by evoking hypoxia, the anti-abnormalization activity of aPlGF may offer promising target to combat HCC.

<div>Abstract<p>Tumor-associated macrophages (TAM) are exposed to multiple microenvironmental cues in tumors, which collaborate to endow these cells with protumoral activities. Hypoxia, caused by an imbalance in oxygen supply and demand because of a poorly organized vasculature, is often a prominent feature in solid tumors. However, to what extent tumor hypoxia regulates the TAM phenotype <i>in vivo</i> is unknown. Here, we show that the myeloid infiltrate in mouse lung carcinoma tumors encompasses two morphologically distinct CD11b<sup>hi</sup>F4/80<sup>hi</sup>Ly6C<sup>lo</sup> TAM subsets, designated as MHC-II<sup>lo</sup> and MHC-II<sup>hi</sup> TAM, both of which were derived from tumor-infiltrating Ly6C<sup>hi</sup> monocytes. MHC-II<sup>lo</sup> TAM express higher levels of prototypical M2 markers and reside in more hypoxic regions. Consequently, MHC-II<sup>lo</sup> TAM contain higher mRNA levels for hypoxia-regulated genes than their MHC-II<sup>hi</sup> counterparts. To assess the <i>in vivo</i> role of hypoxia on these TAM features, cancer cells were inoculated in prolyl hydroxylase domain 2 (<i>PHD2</i>)-haplodeficient mice, resulting in better-oxygenated tumors. Interestingly, reduced tumor hypoxia did not alter the relative abundance of TAM subsets nor their M2 marker expression, but specifically lowered hypoxia-sensitive gene expression and angiogenic activity in the MHC-II<sup>lo</sup> TAM subset. The same observation in PHD2<sup>+/+</sup>→ PHD2<sup>+/−</sup> bone marrow chimeras also suggests organization of a better-oxygenized microenvironment. Together, our results show that hypoxia is not a major driver of TAM subset differentiation, but rather specifically fine-tunes the phenotype of M2-like MHC-II<sup>lo</sup> TAM. <i>Cancer Res; 74(1); 24–30. ©2013 AACR</i>.</p></div>

Epidemiological studies suggest that elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) predispose an individual to ischemic heart disease or promote plaque progression by inhibiting fibrinolysis. In the present study, loss of PAI-1 in apolipoprotein E (apoE)-deficient (apoE −/− :PAI-1 −/− ) mice promoted the growth of advanced atherosclerotic plaques, which was due to enhanced extracellular matrix deposition. ApoE −/− :PAI-1 −/− plaques also exhibited collagen fiber disorganization and degradation. Immunostaining and bone marrow transplantation revealed that smooth muscle cells, not macrophages, primarily expressed PAI-1 in plaques. Thus, although PAI-1 may promote plaque growth because of its antifibrinolytic properties, the present study reveals a protective role for PAI-1 by limiting plaque growth and preventing abnormal matrix remodeling.

The availability of gene-targeted mice deficient in the urokinase-type plasminogen activator (uPA), urokinase receptor (uPAR), tissue-type plasminogen activator (tPA), and plasminogen permits a critical, genetic-based analysis of the physiological and pathological roles of the two mammalian plasminogen activators. We report a comparative study of animals with individual and combined deficits in uPAR and tPA and show that these proteins are complementary fibrinolytic factors in mice. Sinusoidal fibrin deposits are found within the livers of nearly all adult mice examined with a dual deficiency in uPAR and tPA, whereas fibrin deposits are never found in livers collected from animals lacking uPAR and rarely detected in animals lacking tPA alone. This is the first demonstration that uPAR has a physiological role in fibrinolysis. However, uPAR-/-/tPA-/- mice do not develop the pervasive, multi-organ fibrin deposits, severe tissue damage, reduced fertility, and high morbidity and mortality observed in mice with a combined deficiency in tPA and the uPAR ligand, uPA. Furthermore, uPAR-/-/tPA-/- mice do not exhibit the profound impairment in wound repair seen in uPA-/-/tPA-/- mice when they are challenged with a full-thickness skin incision. These results indicate that plasminogen activation focused at the cell surface by uPAR is important in fibrin surveillance in the liver, but that uPA supplies sufficient fibrinolytic potential to clear fibrin deposits from most tissues and support wound healing without the benefit of either uPAR or tPA.

Metabolic reprogramming, a crucial cancer hallmark, shifts metabolic pathways such as glycolysis, tricarboxylic acid cycle or lipogenesis, to enable the growth characteristics of cancer cells. Here, we provide evidence that transketolase-like 1 (TKTL1) orchestrates aerobic glycolysis, fatty acid and nucleic acid synthesis, glutamine metabolism, protection against oxidative stress and cell proliferation. Furthermore, silencing of TKTL1 reduced the levels of sphingolipids such as lactosylceramide (a sphingolipid regulating cell survival, proliferation and angiogenesis) and phosphatidylinositol (which activates PI3K/Akt/mTOR signaling). Thus, in addition to its well-known roles in glucose and amino acid metabolism, TKTL1 also regulates lipid metabolism. In conclusion, our study provides unprecedented evidence that TKTL1 plays central roles in major metabolic processes subject to reprogramming in cancer cells and thus identifies TKTL1 as a promising target for new anti-cancer therapies.

Prominin-1 (CD133) is commonly used to isolate stem and progenitor cells from the developing and adult nervous system and to identify cancer stem cells in brain tumors. However, despite extensive characterization of Prominin-1 + precursor cells from the adult subventricular zone, no information about the expression of Prominin-1 by precursor cells in the subgranular zone (SGZ) of the adult hippocampus has been available. We show here that Prominin-1 is expressed by a significant number of cells in the SGZ of adult mice in vivo and ex vivo , including postmitotic astrocytes. A small subset of Prominin-1 + cells coexpressed the nonspecific precursor cell marker Nestin as well as GFAP and Sox2. Upon fluorescence-activated cell sorting, only Prominin-1/Nestin double-positive cells fulfilled the defining stem cell criteria of proliferation, self-renewal, and multipotentiality as assessed by a neurosphere assay. In addition, isolated primary Prominin-1 + cells preferentially migrated to the neurogenic niche in the SGZ upon transplantation in vivo . Finally, despite its expression by various stem and progenitor cells, Prominin-1 turned out to be dispensable for precursor cell proliferation in vitro and in vivo . Nevertheless, a net decrease in hippocampal neurogenesis, by ∼30% was found in Prominin-1 knock-out mice, suggesting other roles in controlling adult hippocampal neurogenesis. Remarkably, an upregulation of Prominin-2 was detected in Prominin-1-deficient mice highlighting a potential compensatory mechanism, which might explain the lack of severe symptoms in individuals carrying mutations in the Prom1 gene.

Abstract Optimal distribution of heterogeneous organelles and cell types within an organ is essential for physiological processes. Unique for the ovary, hormonally regulated folliculogenesis, ovulation, luteal formation/regression and associated vasculature changes lead to tissue remodeling during each reproductive cycle. Using the CLARITY approach and marker immunostaining, we identified individual follicles and corpora lutea in intact ovaries. Monitoring lifetime changes in follicle populations showed age-dependent decreases in total follicles and percentages of advanced follicles. Follicle development from primordial to preovulatory stage was characterized by 3 × 10 5 -fold increases in volume, decreases in roundness, and decreased clustering of same stage follicles. Construction of follicle-vasculature relationship maps indicated age- and gonadotropin-dependent increases in vasculature and branching surrounding follicles. Heterozygous mutant mice with deletion of hypoxia-response element in the vascular endothelial growth factor A (VEGFA) promoter showed defective ovarian vasculature and decreased ovulatory responses. Unilateral intrabursal injection of axitinib, an inhibitor of VEGF receptors, retarded neo-angiogenesis that was associated with defective ovulation in treated ovaries. Our approach uncovers unique features of ovarian architecture and essential roles of vasculature in organizing follicles to allow future studies on normal and diseased human ovaries. Similar approaches could also reveal roles of neo-angiogenesis during embryonic development and tumorigenesis.

Stabilin‐1 is a transmembrane glycoprotein expressed on activated macrophages and endothelial cells of new capillaries during tissue remodeling. In the coxsackie B3 (CVB3) murine model of viral myocarditis, stabilin‐1 expression increased significantly and was primarily expressed in macrophages. This was also seen in endomyocardial biopsies from patients with fulminant myocarditis, where stabilin‐1 was expressed by CD‐68+ macrophages, forming a protective barrier around the healthy tissue. When challenged with the CVB3 virus, Stabilin‐1 null mice showed increased cardiac related mortality as compared to wild‐type mice (p<0,001, n=19). Histological analysis showed that the stabilin‐1 null mice had increased cardiac necrosis due to CD3+ T‐lymphocyte infiltration, and lower F4/80+ macrophages as compared to their wild‐type littermates, suggesting that stabilin‐1 may be important for monocytes recruitment. Thioglycolate induced peritonitis in stabilin‐1 WT and null mice also resulted in fewer monocytes recruited. Depletion of circulating monocytes with clodronate liposomes in wild‐type mice induced a similar phenotype after CVB3 infection. We identify stabilin‐1 as an important upregulated protein during VM in both mice and humans and that absence of stabilin‐1 results in increased cardiac inflammation, injury and mortality, demonstrating a novel cardio‐protective effect of stabilin‐1.