Showing papers by "Kari Alitalo published in 2022"

The SARS-CoV-2 receptor ACE2 is expressed in mouse pericytes but not endothelial cells: Implications for COVID-19 vascular research

Abstract: Humanized mouse models and mouse-adapted SARS-CoV-2 virus are increasingly used to study COVID-19 pathogenesis, so it is important to learn where the SARS-CoV-2 receptor ACE2 is expressed. Here we mapped ACE2 expression during mouse postnatal development and in adulthood. Pericytes in the CNS, heart, and pancreas express ACE2 strongly, as do perineurial and adrenal fibroblasts, whereas endothelial cells do not at any location analyzed. In a number of other organs, pericytes do not express ACE2, including in the lung where ACE2 instead is expressed in bronchial epithelium and alveolar type II cells. The onset of ACE2 expression is organ specific: in bronchial epithelium already at birth, in brain pericytes before, and in heart pericytes after postnatal day 10.5. Establishing the vascular localization of ACE2 expression is central to correctly interpret data from modeling COVID-19 in the mouse and may shed light on the cause of vascular COVID-19 complications.

Interplay of vascular endothelial growth factor receptors in organ-specific vessel maintenance

Abstract: Using VEGF receptor deletions in mice, the authors show that VEGFR1 and VEGFR3 can support vessel maintenance in the absence of VEGFR2 in an organ-specific manner in postnatal and adult mice.

Lymphangiogenesis requires Ang2/Tie/PI3K signaling for VEGFR3 cell-surface expression

Abstract: Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor 3 (VEGFR3), which is encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development, and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here, we used gene deletion, blocking Abs, transgene induction, and gene transfer to study how Ang2, its Tie2 receptor, and Tie1 regulate lymphatic vessels. We discovered that VEGF-C–induced Ang2 secretion from lymphatic endothelial cells (LECs) was involved in full Akt activation downstream of phosphoinositide 3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of an Ang2-blocking Ab decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of the PI3K catalytic p110α subunit or with small-molecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C–induced lymphangiogenesis also in adult mice. Our results reveal an important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2/Tie/PI3K signaling.

Phase 1 Lymfactin® Study: 24-month Efficacy and Safety Results of Combined Adenoviral VEGF-C and Lymph Node Transfer Treatment for Upper Extremity Lymphedema.

Abstract: Lymphedema is a common problem after breast cancer treatment. Lymfactin® is a prolymphangiogenic growth factor vector inducing the expression of human vascular endothelial growth factor C (VEGF-C). It promotes growth and repair of lymphatic vessels.Lymfactin® was combined with microvascular lymph node transfer surgery (VLNT) to study the safety and efficacy of the treatment in breast cancer-related upper limb lymphedema (BCRL) patients. This is a continuation study with a 3 year efficacy and 5 year safety follow-up.Fifteen patients were recruited in the study between June 2016 and February 2018. Three patients received a lower dose (1 × 1010 viral particles (vp)), and 12 patients received a higher dose (1 × 1011 vp) of Lymfactin®, respectively. In the higher dose group, the reduction of excess arm volume was on average 46% after the 12 month follow-up, and the transport index was improved in 7/12 patients. At baseline, removal of the compression garment for 7 days resulted in significant arm swelling (105.7±161.0 ml, p=0.0253). However, at 12 months, there was less and not significant swelling after removal of the garment (84.4±143.0 ml, p=0.0682). Lymphedema Quality of Life Inventory (LQOLI or LyQLI) questionnaire showed significant and sustained improvement of quality of life.During 24 months' of follow-up, the results indicate that Lymfactin® is well tolerated. The most promising findings were a 46% reduction in excess arm volume and a nonsignificant volume increase after garment removal at 12 months, suggesting that there is potential for the reduction of lymphedema.

Genetic Engineering of Lymphangiogenesis in Skin Does Not Affect Blood Pressure in Mouse Models of Salt-Sensitive Hypertension

Abstract: Background: Recent studies have indicated that sodium storage is influenced by macrophages that secrete VEGF-C (vascular endothelial growth factor) during salt stress thus stimulating lymphangiogenesis, thereby acting as a buffer against increased blood pressure (BP). We aimed to explore the role of dermal lymphatics in BP and sodium homeostasis. Our hypothesis was that mice with reduced dermal lymphatic vessels were more prone to develop salt-sensitive hypertension, and that mice with hyperplastic vessels were protected. Methods: Mice with either hypoplastic (Chy), absent (K14-VEGFR3 [vascular endothelial growth factor receptor 3]-Ig), or hyperplastic (K14-VEGF-C) dermal lymphatic vessels and littermate controls were given high-salt diet (4% NaCl in the chow), deoxycorticosterone acetate (DOCA)-salt diet and 1% saline to drink or nitric oxide blocker diet L-NG-nitro arginine methyl ester (followed by high salt diet). BP was measured by telemetric recording, and tissue sodium content by ion chromatography. Results: In contrast to previous studies, high salt diet did not induce an increase in BP or sodium storage in any of the mouse strains investigated. DOCA-salt, on the other hand, gave an increase in BP in Chy and K14-VEGFR3-Ig not different from their corresponding WT controls. DOCA induced salt storage in skin and muscle, but to the same extent in mice with dysfunctional lymphatic vessels and WT controls. Lymph flow as assessed by tracer washout was not affected by the diet in any of the mouse strains. Conclusions: Our results suggest that dermal lymphatic vessels are not involved in salt storage or blood pressure regulation in these mouse models of salt-sensitive hypertension.

Significance of developmental meningeal lymphatic dysfunction in experimental post-traumatic injury

Abstract: Understanding the pathological mechanisms unfolding after chronic traumatic brain injury (TBI) could reveal new therapeutic entry points. During the post-TBI sequel, the involvement of cerebrospinal fluid drainage through the meningeal lymphatic vessels was proposed. Here, we used K14-VEGFR3-Ig transgenic mice to analyze whether a developmental dysfunction of meningeal lymphatic vessels modifies post-TBI pathology. To this end, a moderate TBI was delivered by controlled cortical injury over the temporal lobe in male transgenic mice or their littermate controls. We performed MRI and a battery of behavioral tests over time to define the post-TBI trajectories. In vivo analyses were integrated by ex-vivo quantitative and morphometric examinations of the cortical lesion and glial cells. In post-TBI K14-VEGFR3-Ig mice, the recovery from motor deficits was protracted compared to littermates. This outcome is coherent with the observed slower hematoma clearance in transgenic mice during the first two weeks post-TBI. No other genotype-related behavioral differences were observed, and the volume of cortical lesions imaged by MRI in vivo, and confirmed by histology ex-vivo, were comparable in both groups. However, at the cellular level, post-TBI K14-VEGFR3-Ig mice exhibited an increased percentage of activated Iba1 microglia in the hippocampus and auditory cortex, areas that are proximal to the lesion. Although not impacting or modifying the structural brain damage and post-TBI behavior, a pre-existing dysfunction of meningeal lymphatic vessels is associated with morphological microglial activation over time, possibly representing a sub-clinical pathological imprint or a vulnerability factor. Our findings suggest that pre-existing mLV deficits could represent a possible risk factor for the overall outcome of TBI pathology.

Endothelial TIE1 restricts angiogenic sprouting to coordinate vein assembly in synergy with its homologue TIE2

Abstract: Objective Vascular growth followed by vessel specification is crucial for the establishment of a hierarchical blood vascular network. We have here investigated mechanisms underlying venogenesis, particularly the molecular control over venous fate acquisition during vascular development. Approach and Results We analyzed the function of TIE1 as well as its synergy with TIE2 in the regulation of vein formation by employing genetic mouse models targeting Tie1 and Tek. Cardinal vein growth appeared normal in TIE1 deficient mice, whereas TIE2 deficiency altered the identity of cardinal vein endothelial cells with the aberrant expression of DLL4. Interestingly, the parallel growth of murine cutaneous veins along with arteries, which was initiated at approximately embryonic day 13.5, was retarded in mice lack of TIE1. Tie1 deletion disrupted also venous integrity, displaying increased sprouting angiogenesis and vascular bleeding. Abnormal venous sprouts with defective arteriovenous alignment were also observed in the mesenteries of Tie1 deleted mice. Mechanistically, TIE1 deficiency resulted in the decreased expression of venous regulators including TIE2 while angiogenic regulators were upregulated. The alteration of TIE2 level by TIE1 insufficiency was further confirmed by the siRNA-mediated knockdown of Tie1 in cultured endothelial cells. Additionally, combining the endothelial deletion of Tie1 with one null allele of Tek resulted in a progressive increase of vein-associated angiogenesis leading to the formation of vascular tufts in retinas, whereas the loss of Tie1 alone produced only a relatively mild venous defect. Conclusions Findings from this study imply that TIE1 and TIE2 act in a synergistic manner to restrict sprouting angiogenesis during vein formation.

PROX1 transcription factor controls rhabdomyosarcoma growth, stemness, myogenic properties and therapeutic targets

Abstract: Significance Rhabdomyosarcoma is an aggressive pediatric soft-tissue cancer. Identification of RMS precursor cells and models has been a challenge due to unknown molecular mechanisms that drive the common proliferative myogenic phenotype. Our study revealed that the PROX1 transcription factor is essential for the growth and myogenic features of RMS, and its downregulation reverts RD cells to a phenotype resembling benign mesenchymal stem cells. Our findings demonstrate that the effects of PROX1 on RMS cell growth are mediated by FGFR, predominantly by FGFR1 and FGFR4, which were found to compensate for each other. This study places PROX1 as a major mediator of RMS characteristics and development and suggests that pan-FGFR inhibitors provide a promising option for the treatment of RMS.

STAT5b is a key effector of NRG-1/ERBB4-mediated cardiomyocyte 1 growth 2 Short title: STAT5b in NRG-1 mediated cardiac growth

Abstract: The growth factor neuregulin-1 (NRG-1) regulates hypertrophic and hyperplastic myocardial growth and is currently under clinical investigation as a treatment for heart failure. We have previously demonstrated that an isoform of the NRG-1 receptor ERBB4 (ERBB4 JM-b) expressed in cardiomyocytes selectively regulates the activation of STAT5b. To explore the 23 role of STAT5b in NRG-1/EBBB4 mediated cardiomyocyte growth, several in vitro and in vivo models were utilized. The downregulation of NRG-1/ERBB4 signaling consistently reduced 25 STAT5b activation and transcription of STAT5b target genes Igf1 , Myc and Cdkn1a in murine 26 in vitro and in vivo models of myocardial growth. Stat5b knock-down in primary 27 cardiomyocytes ablated NRG-1-induced cardiomyocyte hypertrophy. Stat5b was activated during NRG-1-induced hyperplastic myocardial growth and chemical inhibition of the Nrg- 29 1/Erbb4 pathway led to the loss of myocardial growth and Stat5 activation in zebrafish 30 embryos. Moreover, CRISPR/Cas9-mediated knock-down of stat5b in zebrafish embryos 31 resulted in reduced myocardial growth and heart failure as indicated by reduced ventricular 32 ejection fraction. Dynamin-2 was discovered to control the cell surface localization of ERBB4 33 and the chemical inhibition of dynamin-2 downregulated NRG-1/ERBB4/STAT5b signaling in 34 models of hypertrophic and hyperplastic myocardial growth. Finally, the activation of the 35 NRG-1/ERBB4/STAT5b signaling pathway was explored in clinical samples representing 36 pathological cardiac hypertrophy. The NRG-1/ERBB4/STAT5b signaling pathway was differentially regulated both at the mRNA and protein levels in the myocardium of patients with pathological cardiac hypertrophy as compared to myocardium of control subjects. These 39 results establish the role for STAT5b, and dynamin-2 in NRG-1/ERBB4-mediated apoptosis stimulating Ca 2+ indicate a role for NRG-1/ERBB4 signaling in cardiomyocyte apoptosis that cardiomyocyte apoptosis induced dynamin-2 deficiency 42 could be mediated by the loss of NRG-1/ERBB4

Abstract A026: PROX1 transcription factor regulates rhabdomyosarcoma growth, myogenic properties and therapeutic targets

Abstract: Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue cancer with features of skeletal muscle. Because of poor survival of RMS patients and severe long-term side effects of RMS therapies, novel RMS therapies are urgently needed. Here we show that the PROX1 transcription factor is highly expressed in RMS tumors regardless of their cell type of origin. We demonstrate that PROX1 is needed for RMS cell clonogenicity, growth and tumor formation. PROX1 gene silencing repressed several myogenic and tumorigenic transcripts and altered the RD (ERMS) transcriptome to resemble that of benign mesenchymal stem cells. Importantly, we found that fibroblast growth factor receptors (FGFR) mediated the growth effects of PROX1 in RMS. Because of receptor cross-compensation, inhibition of either alone did not mimic the effects of PROX1 silencing, whereas a pan-FGFR inhibitor ablated RMS cell proliferation and decreased tumor xenograft growth. Our findings uncover the critical role of PROX1 in RMS and offer new insights to the mechanisms that regulate RMS development and growth. As FGFR inhibitors have already been tested in unrelated clinical trials, our findings provide a new promising option for RMS treatment. Citation Format: Nebeyu Y. Gizaw, Pauliina Kallio, Erika Gucciardo, Caj Haglund, Tom Bohling, Kaisa Lehti, Mika Sampo, Kari Alitalo, Riikka Kivela. PROX1 transcription factor regulates rhabdomyosarcoma growth, myogenic properties and therapeutic targets [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A026.

β-catenin plus PROX1 immunostaining stratifies disease progression and patient survival in neoadjuvant-treated pancreatic cancer.

Abstract: BACKGROUND Wnt/β-catenin signaling is a highly conserved signaling pathway that regulates the transcription factor PROX1. The role of β-catenin and PROX1 in pancreatic cancer is ambiguous, as some studies have associated their expression with tumor regression and some with tumor progression. OBJECTIVE We have investigated their expression in surgically treated pancreatic cancer patients receiving neoadjuvant therapy (NAT), and patients treated upfront with surgery (US). We furthermore compared the expression of β-catenin and PROX1 between patients who had a good or poor response to NAT. METHODS We evaluated β-catenin and PROX1 expression through immunohistochemistry in 88 neoadjuvant and 144 upfront surgery patients by scoring the intensity of the immunopositivity as 0-3, corresponding to negative, weak, moderate, or strong. We developed a six-tier grading scheme for the neoadjuvant responses by analyzing the remaining tumor cells in surgical specimen histological sections. RESULTS Strong β-catenin immunopositivity associated with improved survival in the patients with good NAT-response (≤10% residual tumor cells) (Hazard ratio [HR] 0.26 95%, confidence interval [CI] 0.07-0.88 p = 0.030). Additionally, the combined moderate β-catenin and PROX1 expression associated with improved survival (HR 0.20 95% CI 0.05-0-76 p = 0.018) among the good responders. Among the patients with a poor NAT-response (> 10% residual tumor cells), both strong β-catenin immunopositivity and strong combined β-catenin and PROX1 associated with shorter survival (HR 2.03 95% CI 1.16-3.55 p = 0.013, and HR 3.1 95% CI 1.08-8.94 p = 0.03, respectively). PROX1 alone was not associated with survival. CONCLUSIONS Strong β-catenin immunopositivity and combined strong or moderate β-catenin and PROX1 immunopositivity associated with improved survival among the good NAT-responders and worse survival among the poor NAT-responders.

Abstract 13155: Apelin and Apela for Therapy of Myocardial Fibrosis and Heart Failure

Abstract: The apelin/apela – apelin receptor system regulates a variety of physiological cardiovascular functions including vascular tone, cardiac contractility, angiogenesis, and energy metabolism. However, the cell type specific effects of apelin/apela in the cardiovascular system are not well understood. The aim of this study was to investigate the potential role of apelin and apela in regulating development of myocardial fibrosis and to identify the signaling mechanisms underlying the anti-fibrotic effects. To investigate the role of apelin and apela in vivo , 2-month-old C57BL/6N male mice were injected with AAV9.LacZ, AAV9.Apelin or AAV9.Apela and subjected to thoracic aortic constriction (TAC) for 6 weeks. Echocardiography analysis at the end of the study showed that overexpression of either apelin or apela attenuated TAC-induced left ventricular (LV) dysfunction and increase in LV mass. Analysis of cardiac tissue showed downregulation of collagen I and fibrosis related periostin mRNA expression levels and reduced accumulation of interstitial fibrosis in the left ventricles of mice overexpressing apelin or apela. Studies in human cardiac fibroblasts in vitro showed that apelin and apela attenuate TGFβ1-induced collagen expression and secretion. Analyses of LV tissue and human fibroblast samples indicated that apelin and apela regulate the stress-induced activation of MAPK pathways. Chemical inhibitors and genetic modification of specific signaling elements identified p38 MAPK as a central signaling pathway mediating the inhibitory effect of apelin and apela on TGFβ1 signaling and TGFβ1-induced collagen production. In summary, we show that apelin and apela attenuate hemodynamic pressure overload -induced LV dysfunction and myocardial fibrosis and identify a novel signaling mechanism regulating the response. Our data suggests that activation of apelinergic signaling could offer a novel therapeutic approach to prevent the development of cardiac fibrosis and heart failure.