Mullistaako sydämen regeneraation mekanismien tutkimus sydäninfarktin jälkeisen lääkehoidon tulevaisuudessa?

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The DAG-responsive C1 domain as a drug target : structure-activity and in vitro pharmacology of isophthalate derivatives

Diacylglycerol (DAG) is a ubiquitous lipid second messenger that transmits signals from cell membrane receptors to intracellular effector proteins containing a specialized DAG recognition motif, the C1 domain. The protein kinase C (PKC) family of serine/threonine kinases is the best-characterized member of DAG effectors, but there are also six other families of proteins with a DAG-responsive C1 domain: (1) the protein kinase D (PKD) family; (2) the myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs); (3) the DAG kinases (DGKs); (4) the Munc13 proteins; (5) the chimaerins; and (6) the Ras guanyl-releasing proteins (RasGRPs). DAG-mediated signalling regulates many cellular functions such as cell proliferation, survival, differentiation, apoptosis and motility − processes that are often deregulated in cancer. As key mediators of these processes, several of the DAG effectors are regarded as promising targets for cancer drug development. Furthermore, PKC contributes to neuronal plasticity and inhibits many pathophysiological processes related to Alzheimer s disease. Activation of PKC is therefore considered a potential future therapeutic strategy for the treatment of Alzheimer s disease. The C1 domain thus represents a well-recognized drug target. However, the existing C1 domain ligands are extremely complex in their chemical structure and from the drug development point of view new C1 domain ligands with a feasible synthesis route are needed. The purpose of these studies was to characterize the structure-activity relationships (SAR) of a novel group of C1 domain ligands, derivatives of 5-(hydroxymethyl)isophthalic acid. These compounds have a simple chemical structure and they are easy to synthesize. Furthermore, these studies aimed to investigate the in vitro pharmacology of the isophthalate derivatives especially focusing on their effects on cell proliferation and morphology. The SAR studies revealed the structural elements indispensable for binding to the C1 domain of PKC: the hydroxymethyl group, both of the ester groups and sufficiently hydrophobic ester substituents. Importantly, the SAR model also held true with β2-chimaerin, another C1-domain containing DAG effector. The active isophthalates bound to PKC with low micromolar affinities, and a selected example of these, HMI-1a3, was also shown to bind to the C1 domains of PKD1 and MRCKα at similar concentrations. Several isophthalates had an antiproliferative effect in HeLa human cervical carcinoma cells. HMI-1a3 exhibited the most potent cytotoxic and antiproliferative effect of the derivatives tested. The isophthalates with no affinity to the C1 domain (e.g. NI-15e, the inactive derivative of HMI-1a3) had no effect on HeLa cell viability or proliferation, suggesting a C1 domain-mediated effect. HMI-1a3 also induced a morphological change characterized by cell elongation. It was accompanied with a marked reorganization of actin cytoskeleton: loss of focal adhesions and actin stress fibres. The elongation-inducing effect of HMI-1a3 was inhibited by an MRCK inhibitor, and thus seems to be at least partially mediated by MRCK. However, the mechanism of its antiproliferative effect remains unclear and may be mediated by several of the DAG effectors. In SH-SY5Y neuroblastoma cells, which are widely used as an in vitro model for neuronal differentiation, HMI-1b11 inhibited cell proliferation and supported neurite growth. HMI-1a3 induced SH-SY5Y cell differentiation as well, but unlike HMI-1b11 it also induced cell death. Again, derivatives with poor binding affinity to the C1 domain had no effect. The HMI-1b11-induced response was accompanied by PKC-dependent ERK1/2 phosphorylation and up-regulation of GAP-43, which is known to mediate neuronal differentiation and to contribute to neurite outgrowth. In conclusion, these studies identify derivatives of 5-(hydroxymethyl)isophthalic acid as a promising novel group of C1 domain ligands. The antiproliferative and cytotoxic HMI-1a3 serves as a potential lead molecule for cancer-related drug discovery. HMI-1b11 on the other hand may be useful in drug development related to neurodegenerative diseases because of its non-toxic and differentiation-inducing properties in SH-SY5Y cells. More studies using cell-based and in vivo models are, however, needed to fully assess the potential of isophthalates in drug development.Toisiolähetti diasyyliglyseroli (DAG) on keskeinen solunsisäisen signaalivälityksen säätelijä. Sen vaikutukset soluissa välittyvät sellaisten proteiinien kautta, joiden rakenne sisältää niin kutsutun C1-osan. Tällaisia proteiineja ovat mm. proteiinikinaasi C (PKC), proteiinikinaasi D (PKD), kimeriinit sekä MRCK (engl. myotonic dystrophy kinase-related Cdc42-binding kinase). DAG:n sitoutuminen kohdeproteiininsa C1-osaan saa aikaan joko proteiinin aktivaation ja/tai muutoksen sen solunsisäisessä sijainnissa. DAG:n säätelemien signaalireittien on havaittu vaikuttavan monien sairauksien, kuten syövän, sydän- ja verisuonitautien ja hermostorappeumasairauksien kehittymiseen. C1-osaa pidetäänkin tästä syystä lupaavana lääkekehityksen kohteena, erityisesti syöpälääkkeiden ja Alzheimerin taudin lääkkeiden kehityksen kannalta. Useimmat tunnetuista C1-osaan sitoutuvista yhdisteistä ovat rakenteeltaan niin monimutkaisia, että niiden kemiallinen synteesi on erittäin vaikeaa ja siten tuottaminen lääkeaineeksi haastavaa. Lääkekehitystä varten tarvitaan siis yksinkertaisempia C1-osaan sitoutuvia yhdisteitä, joiden kemiallinen muokkaus olisi helppoa. Tässä väitöskirjatyössä tutkittiin uusien, kemialliselta rakenteeltaan yksinkertaisten C1-osaan sitoutuviksi suunniteltujen yhdisteiden, isoftalaattijohdannaisten rakenne-vaikutussuhteita ja biologisia vaikutuksia. Työssä selvitettiin C1-osaan sitoutumisen kannalta tärkeät rakenteelliset ominaisuudet isoftalaattirakenteessa tutkimalla yli 40 isoftalaattijohdannaisen sitoutumista PKC:n C1-osaan. PKC:hen sitoutuvien isoftalaattien todettiin sitoutuvan myös β2-kimeriinin, PKD:n ja MRCK:n C1-osiin. Isoftalaateista erityisesti HMI-1a3 esti tehokkaasti kohdunkaulasyöpäsolujen (HeLa-solujen) jakautumista ja sai aikaan solujen muuttumisen pitkänomaisiksi. Solunjakautumisen eston ei kuitenkaan voitu osoittaa välittyvän tietyn yksittäisen DAG:n kohdeproteiinin kautta, ja siksi HMI-1a3 todennäköisesti estää solunjakautumista vaikuttamalla samanaikaisesti useiden C1-osan sisältävien proteiinien toimintaan. HMI-1a3:n aiheuttamaan solujen morfologian muutokseen liittyi huomattavia muutoksia solutukirangassa, ja se näyttäisi välittyvän ainakin osittain MRCK:n kautta, sillä MRCK:n toimintaa estämällä voitiin osittain kumota morfologiassa tapahtuva muutos. Isoftalaattijohdannaisista HMI-1b11 ei vaikuttanut kohdunkaulasyöpäsolujen jakautumiseen, mutta se aiheutti SH-SY5Y-neuroblastoomasolujen erilaistumista enemmän hermosolujen kaltaisiksi soluiksi. Tähän liittyi PKC:n aktivaatio ja mm. oppimisen ja hermosolujen muovautuvuuden kannalta tärkeän GAP-43-proteiinin määrän lisääntyminen. Yhteenvetona voidaan todeta, että dialkyyli 5-(hydroksimetyyli)isoftalaatit ovat lupaava uusi proteiinien C1-osaan sitoutuva yhdisteryhmä, joista HMI-1a3:a voidaan käyttää pohjana syöpälääkkeiden kehityksessä sen syöpäsolujen jakautumista estävän vaikutuksen vuoksi, ja HMI-1b11:lla puolestaan saattaa olla potentiaalia Alzheimerin tautiin liittyvässä lääkekehityksessä. Yhdisteiden vaikutusten tarkemmaksi selvittämiseksi tarvitaan kuitenkin vielä runsaasti sekä solulinjoilla että edellä mainittujen sairauksien eläinmalleissa tehtäviä lisätutkimuksia

Transcriptomics reveal stretched human pluripotent stem cell-derived cardiomyocytes as an advantageous hypertrophy model

Peer reviewe

Distinct Regulation of Cardiac Fibroblast Proliferation and Transdifferentiation by Classical and Novel Protein Kinase C Isoforms : Possible Implications for New Antifibrotic Therapies

Cardiac fibrosis is characterized by accumulation and activation of fibroblasts and excessive production of extracellular matrix, which results in myocardial stiffening and eventually leads to heart failure. Although previous work suggests that protein kinase C (PKC) isoforms play a role in cardiac fibrosis and remodeling, the results are conflicting. Moreover, the potential of targeting PKC with pharmacological tools to inhibit pathologic fibrosis has not been fully evaluated. Here we investigated the effects of selected PKC agonists and inhibitors on cardiac fibroblast (CF) phenotype, proliferation, and gene expression using primary adult mouse CFs, which spontaneously transdifferentiate into myofibroblasts in culture. A 48-hour exposure to the potent PKC activator phorbol 12-myristate 13-acetate (PMA) at 10 nM concentration reduced the intensity of a-smooth muscle actin staining by 56% and periostin mRNA levels by 60% compared with control. The decreases were inhibited with the pan-PKC inhibitor Gö6983 and the inhibitor of classical PKC isoforms Gö6976, suggesting that classical PKCs regulate CF transdifferentiation. PMA also induced a 33% decrease in 5-bromo-2’-deoxyuridine–positive CFs, which was inhibited with Gö6983 but not with Gö6976, indicating that novel PKC isoforms (nPKCs) regulate CF proliferation. Moreover, PMA downregulated the expression of collagen-encoding genes Col1a1 and Col3a1 nPKC-dependently, showing that PKC activation attenuates matrix synthesis in CFs. The partial PKC agonist isophthalate derivative bis(1-ethylpentyl) 5-(hydroxymethyl)isophthalate induced parallel changes in phenotype, cell cycle activity, and gene expression. In conclusion, our results reveal distinct PKC-dependent regulation of CF transdifferentiation and proliferation and suggest that PKC agonists exhibit potential as an antifibrotic treatment.Peer reviewe

Cardiac fibrosis in myocardial infarction - from repair and remodeling to regeneration

Ischemic cell death during a myocardial infarction leads to a multiphase reparative response in which the damaged tissue is replaced with a fibrotic scar produced by fibroblasts and myofibroblasts. This also induces geometrical, biomechanical, and biochemical changes in the uninjured ventricular wall eliciting a reactive remodeling process that includes interstitial and perivascular fibrosis. Although the initial reparative fibrosis is crucial for preventing rupture of the ventricular wall, an exaggerated fibrotic response and reactive fibrosis outside the injured area are detrimental as they lead to progressive impairment of cardiac function and eventually to heart failure. In this review, we summarize current knowledge of the mechanisms of both reparative and reactive cardiac fibrosis in response to myocardial infarction, discuss the potential of inducing cardiac regeneration through direct reprogramming of fibroblasts and myofibroblasts into cardiomyocytes, and review the currently available and potential future therapeutic strategies to inhibit cardiac fibrosis.Peer reviewe

Pharmacological protein kinase C modulators reveal a pro-hypertrophic role for novel protein kinase C isoforms in human induced pluripotent stem cell-derived cardiomyocytes

Background: Hypertrophy of cardiomyocytes (CMs) is initially a compensatory mechanism to cardiac overload, but when prolonged, it leads to maladaptive myocardial remodeling, impairing cardiac function and causing heart failure. A key signaling molecule involved in cardiac hypertrophy is protein kinase C (PKC). However, the role of different PKC isoforms in mediating the hypertrophic response remains controversial. Both classical (cPKC) and novel (nPKC) isoforms have been suggested to play a critical role in rodents, whereas the role of PKC in hypertrophy of human CMs remains to be determined. Here, we aimed to investigate the effects of two different types of PKC activators, the isophthalate derivative HMI-1b11 and bryostatin-1, on CM hypertrophy and to elucidate the role of cPKCs and nPKCs in endothelin-1 (ET-1)-induced hypertrophy in vitro. Methods and Results: We used neonatal rat ventricular myocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study the effects of pharmacological PKC modulators and ET-1. We used quantitative reverse transcription PCR to quantify hypertrophic gene expression and high-content analysis (HCA) to investigate CM morphology. In both cell types, ET-1, PKC activation (bryostatin-1 and HMI-1b11) and inhibition of cPKCs (Gö6976) increased hypertrophic gene expression. In NRVMs, these treatments also induced a hypertrophic phenotype as measured by increased recognition, intensity and area of α-actinin and F-actin fibers. Inhibition of all PKC isoforms with Gö6983 inhibited PKC agonist-induced hypertrophy, but could not fully block ET-1-induced hypertrophy. The mitogen-activated kinase kinase 1/2 inhibitor U0126 inhibited PKC agonist-induced hypertrophy fully and ET-1-induced hypertrophy partially. While ET-1 induced a clear increase in the percentage of pro-B-type natriuretic peptide-positive hiPSC-CMs, none of the phenotypic parameters used in HCA directly correlated with gene expression changes or with phenotypic changes observed in NRVMs. Conclusions: This work shows similar hypertrophic responses to PKC modulators in NRVMs and hiPSC-CMs. Pharmacological PKC activation induces CM hypertrophy via activation of novel PKC isoforms. This pro-hypertrophic effect of PKC activators should be considered when developing PKC-targeted compounds for e.g. cancer or Alzheimer’s disease. Furthermore, this study provides further evidence on distinct PKC-independent mechanisms of ET-1-induced hypertrophy both in NRVMs and hiPSC-CMs.Peer reviewe

Cell adhesion and proliferation on common 3D printing materials used in stereolithography of microfluidic devices

Three-dimensional (3D) printing has recently emerged as a cost-effective alternative for rapid prototyping of microfluidic devices. The feature resolution of stereolithography-based 3D printing is particularly well suited for manufacturing of continuous flow cell culture platforms. Poor cell adhesion or material-induced cell death may, however, limit the introduction of new materials to microfluidic cell culture. In this work, we characterized four commercially available materials commonly used in stereolithography-based 3D printing with respect to long-term (2 month) cell survival on native 3D printed surfaces. Cell proliferation rates, along with material-induced effects on apoptosis and cell survival, were examined in mouse embryonic fibroblasts. Additionally, the feasibility of Dental SG (material with the most favored properties) for culturing of human hepatocytes and human-induced pluripotent stem cells was evaluated. The strength of cell adhesion to Dental SG was further examined over a shear force gradient of 1–89 dyne per cm² by using a custom-designed microfluidic shear force assay incorporating a 3D printed, tilted and tapered microchannel sealed with a polydimethylsiloxane lid. According to our results, autoclavation of the devices prior to cell seeding played the most important role in facilitating long-term cell survival on the native 3D printed surfaces with the shear force threshold in the range of 3–8 dyne per cm².Three-dimensional (3D) printing has recently emerged as a cost-effective alternative for rapid prototyping of microfluidic devices. The feature resolution of stereolithography-based 3D printing is particularly well suited for manufacturing of continuous flow cell culture platforms. Poor cell adhesion or material-induced cell death may, however, limit the introduction of new materials to microfluidic cell culture. In this work, we characterized four commercially available materials commonly used in stereolithography-based 3D printing with respect to long-term (2 month) cell survival on native 3D printed surfaces. Cell proliferation rates, along with material-induced effects on apoptosis and cell survival, were examined in mouse embryonic fibroblasts. Additionally, the feasibility of Dental SG (material with the most favored properties) for culturing of human hepatocytes and human-induced pluripotent stem cells was evaluated. The strength of cell adhesion to Dental SG was further examined over a shear force gradient of 1-89 dyne per cm(2)by using a custom-designed microfluidic shear force assay incorporating a 3D printed, tilted and tapered microchannel sealed with a polydimethylsiloxane lid. According to our results, autoclavation of the devices prior to cell seeding played the most important role in facilitating long-term cell survival on the native 3D printed surfaces with the shear force threshold in the range of 3-8 dyne per cm(2).Peer reviewe

Application of Human Induced Pluripotent Stem Cell Technology for Cardiovascular Regenerative Pharmacology

Cardiovascular diseases are one of the leading causes of mortality in the western world. Myocardial infarction is among the most prevalent and results in significant cell loss within the myocardium. Similarly, numerous drugs have been identified as having cardiotoxic side effects. The adult human heart is however unable to instigate an effective repair mechanism and regenerate the myocardium in response to such damage. This is in large part due to the withdrawal of cardiomyocytes (CMs) from the cell cycle. Thus, identifying, screening, and developing agents that could enhance the proliferative capacity of CMs holds great potential in cardiac regeneration. Human induced pluripotent stem cells (hiPSCs) and their cardiovascular derivatives are excellent tools in the search for such agents. This chapter outlines state-of-the art techniques for the two-dimensional differentiation and attainment of hiPSC-derived CMs and endothelial cells (ECs). Bioreactor systems and three-dimensional spheroids derived from hiPSC-cardiovascular derivatives are explored as platforms for drug discovery before focusing on relevant assays that can be employed to assess cell proliferation and viability.Peer reviewe

Protein Kinase A-Mediated Effects of Protein Kinase C Partial Agonist 5-(Hydroxymethyl)Isophthalate 1a3 in Colorectal Cancer Cells

Colorectal cancer is the third most commonly occurring cancer in men and the second in women. The global burden of colorectal cancer is projected to increase to over 2 million new cases with over 1 million deaths within the next 10 years and there is a great need for new compounds with novel mechanisms of action. Our group has developed PKC modulating isophthalic acid derivatives that induce cytotoxicity towards human cervical and prostate cancer cell lines. In this study, we investigated the effects of 5-(hydroxymethyl)isophthalate 1a3 (HMI-1a3) on colorectal cancer cell lines (Caco2, Colo205 and HT29). HMI-1a3 inhibited cell proliferation, decreased cell viability and induced an apoptotic response in all studied cell lines. These effects, however, were independent of PKC. Using serine/threonine kinome profiling and pharmacological kinase inhibitors we identified activation of the cAMP/PKA pathway as a new mechanism-of-action for HMI-1a3-induced anti-cancer activity in colorectal cancer cell lines. Our current results strengthen the hypothesis for HMI-1a3 as a potential anti-cancer agent against various malignancies. Significance Statement Colorectal cancer (CRC) is a common solid organ malignancy. Here, we demonstrate that the protein kinase C (PKC) C1 domain-targeted isophthalatic acid derivative HMI-1a3 has anti-cancer activity on CRC cell lines independently of PKC. We identified protein kinase A (PKA) activation as a mechanism of HMI-1a3 induced anti-cancer effects. Our results reveal a new anti-cancer mechanism of action for the partial PKC agonist HMI-1a3 and thus provide new insights for the development of PKC and PKA modulators for cancer therapy.Peer reviewe

Anticancer activity of the protein kinase C modulator HMI-1a3 in 2D and 3D cell culture models of androgen-responsive and androgen-unresponsive prostate cancer

Prostate cancer is one of the most common cancers in men. Although it has a relatively high 5-year survival rate, development of resistance to standard androgen-deprivation therapy is a significant clinical problem. Therefore, novel therapeutic strategies are urgently needed. The protein kinase C (PKC) family is a putative prostate cancer drug target, but so far no PKC-targeting drugs are available for clinical use. By contrast to the standard approach of developing PKC inhibitors, we have developed isophthalate derivatives as PKC agonists. In this study, we have characterized the effects of the most potent isophthalate, 5-(hydroxymethyl) isophthalate 1a3 (HMI-1a3), on three prostate cancer cell lines (LNCaP, DU145, and PC3) using both 2D and 3D cell culture models. In 2D cell culture, HMI-1a3 reduced cell viability or proliferation in all cell lines as determined by the metabolic activity of the cells (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay) and thymidine incorporation. However, the mechanism of action in LNCaP cells was different to that in DU145 or PC3 cells. In LNCaP cells, HMI-1a3 induced a PKC-dependent activation of caspase 3/7, indicating an apoptotic response, whereas in DU145 and PC3 cells, it induced senescence, which was independent of PKC. This was observed as typical senescent morphology, increased beta-galactosidase activity, and upregulation of the senescence marker p21 and downregulation of E2F transcription factor 1. Using a multicellular spheroid model, we further showed that HMI-1a3 affects the growth of LNCaP and DU145 cells in a 3D culture, emphasizing its potential as a lead compound for cancer drug development.Peer reviewe