A patient with intractable pain on high dose opioid therapy. Could we manage not to escalate the opioid dose?

Prolonged opioid treatment reveals problems, like opioid tolerance and opioid induced hyperalgesia. On every stage of disease it should be remembered to use procedures that can have opioid dose sparing effect. We describe a patient with severe mixed neuropathic and nociceptive pain who despite complex medication embracing high dose of morphine suffered from untractable pain. He responded to opioid antagonist with sequential opioid rotation and a simple minimally invasive procedure.Prolonged opioid treatment reveals problems, like opioid tolerance and opioid induced hyperalgesia. On every stage of disease it should be remembered to use procedures that can have opioid dose sparing effect. We describe a patient with severe mixed neuropathic and nociceptive pain who despite complex medication embracing high dose of morphine suffered from untractable pain. He responded to opioid antagonist with sequential opioid rotation and a simple minimally invasive procedure

Understanding PI3K signalling in vessel growth and pathophysiology

[eng] Our knowledge on the molecular basis behind physiological angiogenesis has significantly expanded in the last two decades. This progress also led to improvement in understanding of many vascular-related diseases in which angiogenesis is pathologically altered. Among many molecular regulators of angiogenesis, a family of lipid kinases called phosphatidylinositol 3-kinases (PI3Ks) occupies an important position in controlling endothelial cell functions. Indeed, numerous studies showed that endothelial cells are highly sensitive to fluctuations in the levels of phospholipids generated by these enzymes. Although highly conservative, the eight PI3K isoforms can produce three different types of phospholipids and this phenomenon formed the basis of the division into three different classes. Two members, ass I PI3Kα and ass II PI3K-C2α, are essential for proper vascular development. Moreover, somatic activating mutations in the gene encoding PI3Kα (PIK3CA) were found to cause 25% of venous malformations – a non-malignant, painful and mainly pediatric vascular disease for which the treatment options are limited. The vascular function of other isoforms, in particular class II PI3K-C2β, remains enigmatic. This is surprising given that this isoform is also express in cultured endothelial cells. This thesis is composed of two principal objectives objectives which together have been conceived to increase our knowledge on PI3K signaling in the endothelium. In the first part I evaluated the therapeutic efficacy of pan-AKT inhibitor, miransertib, in Pik3ca-driven vascular malformations using a preclinical mouse model. I showed that miransertib significantly prevents and reverts Pik3ca associated vascular hyperplasia through inhibition of endothelial cell proliferation. My results provide rationale for the therapeutic intervention of miransertib in treating patients with vascular malformations. The second part of the thesis studies the impact of PI3K-C2β isoform on blood vessel expansion and endothelial cell biology. Using both in vivo and in vitro models, I demonstrated for the first time that PI3K-C2β regulates retinal vascularity and vessel width, most likely as result of elevated vascular mTORC1 activity. Moreover, PI3K-C2β kinase inactivation led to increased collagen IV deposition and more stable vascular connections. In parallel, we showed that blood vessel-associated pericytes express high levels of PI3K-C2β and that its loss of function alters their morphology. Finally, we addressed the role of PI3K-C2β in the pathological neoangiogenesis associated with oxygen-induced retinopathy

Understanding PI3K signalling in vessel growth and pathophysiology

Programa de Doctorat en Biomedicina[eng] Our knowledge on the molecular basis behind physiological angiogenesis has significantly expanded in the last two decades. This progress also led to improvement in understanding of many vascular-related diseases in which angiogenesis is pathologically altered. Among many molecular regulators of angiogenesis, a family of lipid kinases called phosphatidylinositol 3-kinases (PI3Ks) occupies an important position in controlling endothelial cell functions. Indeed, numerous studies showed that endothelial cells are highly sensitive to fluctuations in the levels of phospholipids generated by these enzymes. Although highly conservative, the eight PI3K isoforms can produce three different types of phospholipids and this phenomenon formed the basis of the division into three different classes. Two members, ass I PI3Kα and ass II PI3K-C2α, are essential for proper vascular development. Moreover, somatic activating mutations in the gene encoding PI3Kα (PIK3CA) were found to cause 25% of venous malformations – a non-malignant, painful and mainly pediatric vascular disease for which the treatment options are limited. The vascular function of other isoforms, in particular class II PI3K-C2β, remains enigmatic. This is surprising given that this isoform is also express in cultured endothelial cells. This thesis is composed of two principal objectives objectives which together have been conceived to increase our knowledge on PI3K signaling in the endothelium. In the first part I evaluated the therapeutic efficacy of pan-AKT inhibitor, miransertib, in Pik3ca-driven vascular malformations using a preclinical mouse model. I showed that miransertib significantly prevents and reverts Pik3ca associated vascular hyperplasia through inhibition of endothelial cell proliferation. My results provide rationale for the therapeutic intervention of miransertib in treating patients with vascular malformations. The second part of the thesis studies the impact of PI3K-C2β isoform on blood vessel expansion and endothelial cell biology. Using both in vivo and in vitro models, I demonstrated for the first time that PI3K-C2β regulates retinal vascularity and vessel width, most likely as result of elevated vascular mTORC1 activity. Moreover, PI3K-C2β kinase inactivation led to increased collagen IV deposition and more stable vascular connections. In parallel, we showed that blood vessel-associated pericytes express high levels of PI3K-C2β and that its loss of function alters their morphology. Finally, we addressed the role of PI3K-C2β in the pathological neoangiogenesis associated with oxygen-induced retinopathy

Phosphoinositide 3-Kinase-Regulated pericyte maturation governs vascular remodeling

© 2020 American Heart Association, Inc.Background: Pericytes regulate vessel stabilization and function, and their loss is associated with diseases such as diabetic retinopathy or cancer. Despite their physiological importance, pericyte function and molecular regulation during angiogenesis remain poorly understood. Methods: To decipher the transcriptomic programs of pericytes during angiogenesis, we crossed Pdgfrb(BAC)-CreERT2 mice into RiboTagflox/flox mice. Pericyte morphological changes were assessed in mural cell-specific R26-mTmG reporter mice, in which low doses of tamoxifen allowed labeling of single-cell pericytes at high resolution. To study the role of phosphoinositide 3-kinase (PI3K) signaling in pericyte biology during angiogenesis, we used genetic mouse models that allow selective inactivation of PI3Kα and PI3Kβ isoforms and their negative regulator phosphate and tensin homolog deleted on chromosome 10 (PTEN) in mural cells. Results: At the onset of angiogenesis, pericytes exhibit molecular traits of cell proliferation and activated PI3K signaling, whereas during vascular remodeling, pericytes upregulate genes involved in mature pericyte cell function, together with a remarkable decrease in PI3K signaling. Immature pericytes showed stellate shape and high proliferation, and mature pericytes were quiescent and elongated. Unexpectedly, we demonstrate that PI3Kβ, but not PI3Kα, regulates pericyte proliferation and maturation during vessel formation. Genetic PI3Kβ inactivation in pericytes triggered early pericyte maturation. Conversely, unleashing PI3K signaling by means of PTEN deletion delayed pericyte maturation. Pericyte maturation was necessary to undergo vessel remodeling during angiogenesis. Conclusions: Our results identify new molecular and morphological traits associated with pericyte maturation and uncover PI3Kβ activity as a checkpoint to ensure appropriate vessel formation. In turn, our results may open new therapeutic opportunities to regulate angiogenesis in pathological processes through the manipulation of pericyte PI3Kβ activity.Dr Graupera’s laboratory is supported by the research grants SAF2017-89116R-P from Ministerio de Ciencia (Spain) cofunded by European Regional Developmental Fund (ERDF), a Way to Build Europe; by the Catalan government through the project 2017-SGR; by La Caixa Foundation (HR18-00120); by la Asociación Española contra el Cancer (AECC)-Grupos Traslacionales (GCTRA18006CARR); by la Fundación BBVA (Beca Leonardo a Investigadores y Creadores Culturales 2017); and by the People Program (Marie Curie Actions; grant agreement 317250) of the European Union’s Seventh Framework Program FP7/2007 to 2013/, and the Marie Skłodowska-Curie (grant agreement 675392) of the European Union’s Horizon 2020 research. Dr Carracedo’s laboratory is supported by the Basque Department of Industry, Tourism and Trade (Elkartek) and the Department of Education (IKERTALDE IT1106-16), the Ministerio de Ciencia (SAF2016-79381-R [FEDER/EU], Severo Ochoa Excellence Accreditation SEV-2016-0644; Excellence Networks SAF2016-81975-REDT), European Training Networks Project (H2020-MSCA-ITN-308 2016 721532), the AECC (IDEAS175CARR, GCTRA18006CARR), La Caixa Foundation (HR17-00094), and the European Research Council (StG 336343, PoC 754627, CoG 819242). Centro de Investigación Biomédica en Red Cáncer (CIBERONC) was cofunded with FEDER funds and funded by Instituto de Salud Carlos III. Dr Aransay’s laboratory is supported by the Basque Department of Industry, Tourism and Trade (Elkartek) and the Severo Ochoa Excellence Accreditation SEV-2016-0644. Dr Franco was supported by European Research Council (StG 679368), the H2020-Twinning grant (692322), the Fundação para a Ciência e a Tecnologia funding (grants IF/00412/2012; EXPL-BEX-BCM-2258-2013; PRECISE-LISBOA-01-0145-FEDER-016394), and a grant from the Fondation Leducq (17CVD03). Personal support was from Marie-Curie ITN Actions (Dr Figueiredo and Kobialka), Juan de la Cierva (IJCI-2015-23455, Dr Villacampa), and CIBERONC (A. Martinez-Romero).info:eu-repo/semantics/publishedVersio

The onset of PI3K-related vascular malformations occurs during angiogenesis and is prevented by the AKT inhibitor miransertib

Low-flow vascular malformations are congenital overgrowths composed of abnormal blood vessels potentially causing pain, bleeding and obstruction of different organs. These diseases are caused by oncogenic mutations in the endothelium, which result in overactivation of the PI3K/AKT pathway. Lack of robust in vivo preclinical data has prevented the development and translation into clinical trials of specific molecular therapies for these diseases. Here, we demonstrate that the Pik3caH1047R activating mutation in endothelial cells triggers a transcriptome rewiring that leads to enhanced cell proliferation. We describe a new reproducible preclinical in vivo model of PI3K-driven vascular malformations using the postnatal mouse retina. We show that active angiogenesis is required for the pathogenesis of vascular malformations caused by activating Pik3ca mutations. Using this model, we demonstrate that the AKT inhibitor miransertib both prevents and induces the regression of PI3K-driven vascular malformations. We confirmed the efficacy of miransertib in isolated human endothelial cells with genotypes spanning most of human low-flow vascular malformations.The research leading to these results has received funding by the Spanish Ministry of Science and Innovation MICINN (PID2020-116184RB-I00 /AEI/10.13038/501100011033). M.G. laboratory is supported by the research grants SAF2017-89116R-P (FEDER/EU) from MCIU (Spain) co-funded by European Regional Developmental Fund (ERDF), a Way to Build Europe and PID2020-116184RB-I00 from MCEI; PTEN RESEARCH Foundation (IJC-21-001); la Caixa Banking Foundation (LCF/PR/PR16/51110035 and LCF/PR/HR19/52160023; also to E.B. and J.M.); by la Asociación Española contra el Cancer (AECC)-Grupos Traslacionales (GCTRA18006CARR); by la Fundación BBVA (Ayuda Fundación BBVA a Equipos de Investigación Científica 2019); World Cancer Research (21-0159). The computations and data handling were enabled by resources in projects SNIC 2019/30-26 and SNIC 2019/8-129, provided by the Swedish National Infrastructure for Computing (SNIC) at UPPMAX, partially funded by the Swedish Research Council through grant agreement no. 2018-05973. Personal support was from Marie-Curie ITN Actions (P.K. and J.Z.) grant agreement 675392. L.G. is funded by the Swedish Research Council (2018-06591). S.D.C. is a recipient of a fellowship from the European Union's Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement No 749731. S.D.C. is currently funded by la Caixa Banking Foundation Junior Leader project (LCF/BQ/PR20/11770002). E.B. is funded by the Agencia Estatal de Investigación (Proyectos de investigación en salud PI20/00102)

Endothelial cell rearrangements during vascular patterning require PI3-kinase-mediated inhibition of actomyosin contractility

Angiogenesis is a dynamic process relying on endothelial cell rearrangements within vascular tubes, yet the underlying mechanisms and functional relevance are poorly understood. Here we show that PI3Kα regulates endothelial cell rearrangements using a combination of a PI3Kα-selective inhibitor and endothelial-specific genetic deletion to abrogate PI3Kα activity during vessel development. Quantitative phosphoproteomics together with detailed cell biology analyses in vivo and in vitro reveal that PI3K signalling prevents NUAK1-dependent phosphorylation of the myosin phosphatase targeting-1 (MYPT1) protein, thereby allowing myosin light chain phosphatase (MLCP) activity and ultimately downregulating actomyosin contractility. Decreased PI3K activity enhances actomyosin contractility and impairs junctional remodelling and stabilization. This leads to overstretched endothelial cells that fail to anastomose properly and form aberrant superimposed layers within the vasculature. Our findings define the PI3K/NUAK1/MYPT1/MLCP axis as a critical pathway to regulate actomyosin contractility in endothelial cells, supporting vascular patterning and expansion through the control of cell rearrangement

Endothelial cell rearrangements during vascular patterning require PI3-kinase-mediated inhibition of actomyosin contractility

Angiogenesis is a dynamic process relying on endothelial cell rearrangements within vascular tubes, yet the underlying mechanisms and functional relevance are poorly understood. Here we show that PI3Kα regulates endothelial cell rearrangements using a combination of a PI3Kα-selective inhibitor and endothelial-specific genetic deletion to abrogate PI3Kα activity during vessel development. Quantitative phosphoproteomics together with detailed cell biology analyses in vivo and in vitro reveal that PI3K signalling prevents NUAK1-dependent phosphorylation of the myosin phosphatase targeting-1 (MYPT1) protein, thereby allowing myosin light chain phosphatase (MLCP) activity and ultimately downregulating actomyosin contractility. Decreased PI3K activity enhances actomyosin contractility and impairs junctional remodelling and stabilization. This leads to overstretched endothelial cells that fail to anastomose properly and form aberrant superimposed layers within the vasculature. Our findings define the PI3K/NUAK1/MYPT1/MLCP axis as a critical pathway to regulate actomyosin contractility in endothelial cells, supporting vascular patterning and expansion through the control of cell rearrangement