Metalliproteinaasit demyelinisoivassa taudissa - Luento

http://www.youtube.com/watch?v=JGMEjdhWZTw

Elastaasi, ihmisen elastaasigeenit. MMP-12 on makrofagielastaasi.

Päivitystä 17.4. 2014. 
WIKIPEDIA kertoo, että ihmisellä on kahdeksan elastaasigeeniä. ( Tämä artikkeli on keskeneräinen)
Mikä näistä olisi kyseessä aortan aneurysmissa?
 ( Ainakin MMP-9 erään lähteen mukaan. http://www.chisholm.northwestern.edu/chisholm/MMP9.htm )

Wikipedialähde maintisee että myös MMP-12 osallistuu aneurysman muodostukseen. (MMP12 may play a role in aneurysm formation^[4] and studies in mice and humans suggest a role in the development of emphysema).

 http://en.wikipedia.org/wiki/Neutrophil_elastase

Interactions Neutrophil elastase has been shown to interact with Alpha 2-antiplasmin.

There exist eight human genes for elastase:

Family	Gene symbol		Protein name		EC number
	Approved	Previous	Approved	Previous
chymotrypsin- like	CELA1	ELA1	chymotrypsin-like elastase family, member 1	elastase 1, pancreatic	EC 3.4.21.36
	CELA2A	ELA2A	chymotrypsin-like elastase family, member 2A	elastase 2A, pancreatic	EC 3.4.21.71
	CELA2B	ELA2B	chymotrypsin-like elastase family, member 2B	elastase 2B, pancreatic	EC 3.4.21.71
	CELA3A	ELA3A	chymotrypsin-like elastase family, member 3A	elastase 3A, pancreatic	EC 3.4.21.70
	CELA3B	ELA3B	chymotrypsin-like elastase family, member 3B	elastase 3B, pancreatic	EC 3.4.21.70
chymotrypsin	CTRC	ELA4	chymotrypsin C (caldecrin)	elastase 4	EC 3.4.21.2
neutrophil	ELANE	ELA2	neutrophil elastase	elastase 2	EC 3.4.21.37
macrophage	MMP12	HME	macrophage metalloelastase	macrophage elastase	EC 3.4.24.65

Amiloridi

Amiloridi
3,5-diamino-6-chloro-N-(diaminomethylene)pyrazine-2-carboxamide
 on kaliumia säästävä diureettinen lääke.

 Sitä on alettu kokeilla 1967 hypertension hoitoon ja lievässä sydäninsuffisienssissa. (Itsekin olen kirjoittanut tätä lääkettä Moduretic nimisenä, jossa se esiintyy kombinaationa tiatsidiryhmän diureetin kanssa) Amiloridissa on guanidiiniryhmä pyratsiinijohdannaisena.

 Amiloridi pystyy suoraan blokeeraamaan epiteliaalisen natriumkanavan (ANaC) ja täten estämään natriumin reabsorption samaan tapaan kuin triamtereeni, tästä seuraa natriumin ja veden menetystä ja kaliumin samanaikaista säästöä.- usein kombinoidaan amiloridi tiatsidiin ( Moduretic) tai furesikseen , loop- diuretikaan

. Joskus voi kehittyä hyperkalemiaa amiloridissta. Riski on suuri jos käytetään ACE -estäjiä tai spironolaktonia samaan aikaan.

 Samalla mainitaan ettei tulisi käyttää kaliumpitoisia pöytäsuoloja ( Seltin ym)

Amiloridilla on myös asidoosin kehittämisriskinsä.
 Amiloridin eräs vaikutus on cGMP-toimivan kationikanavna estäminen munuaisen ytimen sisemmässä kokoojatiehyessä ( inner medullary collecting duct.)

 Sydänvaikutuksenaan amiloridi sekundäärisesti blokeeraa Na+/H+ vaihtajan(NHE-1) Tämä minimoi iskemisten kohtausten reperfuusion aiheuttamia vaurioita.
Päivitys 18.4. 2014.

Monosyytti ja MMP. Aortta

Haku 16.4. 2014) Lähde:

Samadzadeh KM¹, Chun KC¹ et al.  Monocyte activity is linked with abdominal aortic aneurysm diameter. J Surg Res. 2014 Mar 13. pii: S0022-4804(14)00252-2. doi: 10.1016/j.jss.2014.03.019. [Epub ahead of print]

Tiivistelmä( suomennosta)  Abstract

 

Taustaa: 

• Systeeminen tulehdus ja lisääntynyt matrixmetalloproteinaasien (MMP) määrä aiheuttaa  elastiinin,  aortalle tyypillisen  proteiinirakenteen,  hajoamista, mistä seuraa  vatsa-alueen aortan aneurysman laajenemista. Usea prospektiivinen tutkimus raportoi, että statiinihoidolla voidaan vähentää vatsa-alueen  valtavaltimon aneurysmien laajenemisia  anti-inflammatorisen vaikutuksen välityksellä.  Tutkijat ovat tehneet oletuksensa, että monosyyttien  aktiviisuudella on olennaista osuutta tässä vatsa-alueen  valtavaltimon aneurysman muodostumisessa  ja sen takia tässä tutkimuksessa  selvitetään  potilaan perifeerisen veren monosyyttien  soluadhesiivisuutta, migraatiota  suonen sisäkalvon endoteelin läpi ja matrixmetalloproteinaasientsyymien pitoisuuksia   vatsa-alueen  aorta-aneurysmaa potevilla ja kontrollihenkilöillä, joilla ei ole  aneurysmaa tässä osassa valtavaltimoaan. .

BACKGROUND: Systemic inflammation and increased matrix metalloproteinase (MMP) cause elastin degradation leading to abdominal aortic aneurysm (AAA) expansion. Several prospective studies report that statin therapy can reduce AAA expansion through anti-inflammation. We hypothesize that monocyte activity plays a pivotal role in this AAA development and this study examines patient peripheral blood monocyte cell adhesion, transendothelial migration, and MMP concentrations between AAA and non-AAA patients.

•  Tutkimusaineisto ja menetelmät. Perifeerisen veren näytteitä ja verestä eristettyjä monosyyttejä  viideltätoista terveeltä kontrollilta ja 13 aneurysmapotilaalta. Mitattiin monosyyttein adheesio ja  transmigraatiokyky sekä permeabiliteetti. Luminexmenetelmällä määritettiin MMP-9 ( matrixmetalloproteiinin) ja TIMP- 4 ( metalloproteinaasin kudosestäjän)  pitoisuudet soluviljelmän supernatantista ja potilaan seerumista.

MATERIALS AND METHODS: Peripheral blood was collected and monocytes isolated from control (n = 15) and AAA (n = 13) patients. Monocyte adhesion, transmigration, and permeability assays were assessed. Luminex assays determined MMP-9 and tissue inhibitor of metalloproteinase-4 (TIMP-4) concentrations from cell culture supernatant and patient serum.

 Minkälaisia tulokset olivat ?  Abdominaalialueen aorttaa potevilla oli  monosyyttien adhesoituminen( takertuminen)  suonen sisäpintaan (endoteeliin)  kohonnutta kontrollihenkilöihin verraten. Monosyyttien kulkeminen  kalvon läpio oli myös lisääntynyttä aneyrysmaisilla potilailla. Takertuvien ja transmigroituvien  monosyyttien suurempi lukumäärä oli suoraan verrannollinen vatsa-alueen aortan aneurysman  läpimittaan.Merkitsevästi  korkempi MMP_9 metalloproteinaasin pitoisuus havaittiin aneurysmaisten joukossa verrattuna kontrolleihin. Aneurysmaisten  estäjäentsyymien TIMP-4 pitoisuudet vastaavasti olivat myös merkitsevästi  matalammat  kuin kontrolleilla. Solujen samanaikaisviljelmien supernatanteista mitatut MMP ja TIMP-pitoisuudet  olivat korkeammat kuin pelkkien monosyyttin viljelmistä mitatut pitoisuudet.

RESULTS: AAA patient monocytes showed increased adhesion to the endothelium relative fluorescence units (RFU, 0.33 ± 0.17) versus controls (RFU, 0.13 ± 0.04; P = 0.005). Monocyte transmigration was also increased in AAA patients (RFU, 0.33 ± 0.11) compared with controls (RFU, 0.25 ± 0.04, P = 0.01). Greater numbers of adhesive (R² = 0.66) and transmigratory (R² = 0.86) monocytes were directly proportional to the AAA diameter. Significantly higher serum levels of MMP-9 (2149.14 ± 947 pg/mL) were found in AAA patients compared with controls (1189.2 ± 293; P = 0.01). TIMP-4 concentrations were significantly lower in AAA patients (826.7 ± 100 pg/mL) compared with controls (1233 ± 222 pg/mL; P = 0.02). Cell culture supernatant concentrations of MMP and TIMP from cocultures were higher than monocyte-only cultures.

• Mitä tuli johtopäätökseksi? 

Vatsa-alueen aortan aneurysmaa potevilla henkilöillä monosyytit olivat takertuvaisempia ja ne siirtyivät helpommin suonen endoteelin  läpi koeputkessa tarkastellen ja siitä johtui  kohonneita MMP-9 pitoisuuksia  ja (- loogisesti tähän sopien- ) alentuneita  TIMP-4 pitoisuuksia. Jos pystyttäisiin  moduloimaan monosyyttien aktiivisuutta, voitaisiin   löytää uusia lääketieteellisiä  terapiamahdollisuuksia, joilla saataisiin  vähennettyä  vatsa-alueen aortan aneurysman laajenemista.

CONCLUSIONS: Monocytes from AAA patients have greater adhesion and transmigration through the endothelium in vitro, leading to elevated MMP-9 levels and the appropriate decrease in TIMP-4 levels. The ability to modulate monocyte activity may lead to novel medical therapies to decrease AAA expansion.

Copyright © 2014 Elsevier Inc. All rights reserved.

KEYWORDS:

Abdominal aortic aneurysm, Adhesion, Inflammation, Matrix metalloproteinase, Monocytes, Transmigration

Hepatoman metastasoituminen .Solukalvoreseptori CD147

LÄHDE (2006) : 

 http://cat.inist.fr/?aModele=afficheN&cpsidt=17992653

LÄHDE:  YU Xiao-Ling et al. The glycosylation characteristic of hepatoma-associated antigen HAbl8G/CD147 in human hepatoma cells

Eräs hyvin glykosyloitunut transmembraaninen proteiini (HAbl8G/CD147 ) kuuluu immunoglobuliinien superperheeseen. Tutkijaryhmä oli aiemmin osoittanut, että tämän antigeenin ylimääräinen esiintymä lisäsi ihmisen maksasyöpäsolujen metastaattista potentiaalia.

 

Tässä tutkimuksessa keskityttiin selvittämään ihmisen maksasoluissa tämän Ig-proteiinin glykosyloitumisen piirteet.

 Tätä Ig molekyyliä  eristettiin ihmisen maksasoluista ja sitä  johdettiin ihmisen fibroblastisoluihin, joissa se indusoi matrixmetalloproteinaaseja (MMPs).

Fibroblastin erittämät MMP-entsyymit tutkittiin gelatiinisymografialla.

Hepatomasoluista löydettiin kaksi päämuotoa tätä Ig:tä. 

Selvitettiin, mikä vaikutti eri molekyylikoot, olisiko se glykosylaatio? 

Tässä tutkimuksessa käytettiin tekniikkana deglykosylaatiota, jossa tunicamysiini esti N-glykosylaation tai sitten käytettiin endoglykosidaaseja. Molemmillä metodeilla löydettiin ydinjakso, joka oli kDa 27 kokoa.

Endoglykosidaaseilla selvitettiin,että oligosakkaridiketjutyypit olivat erilaisia ja täten eri endoglykosidaaseille herkkiä.

Johtopäätöksenä oli, että ao. maksasyöpäsolun immunoglobuliini omasi ihmisen fibroblasteihin bioaktiivisuutta stimuloiden niitä tuottamaan kohonneita määriä matrixmetalloproteinaaseja (MMPs).

Lisäksi todettiin että ihmisen maksasyöpäsoluissa on kahta erilaista antigeenia (HAb 18G/CD147), immunoglobuliinimuotoa, joiden ydinosa oli samanlainen, mutta glykosylaatioitten asteet ja tyypit erosivat.

Avainsanat. 

HAb18G/CD147; Glycosylation ; Hepatoma cell ; Endoglycosidase ; Matrix metalloproteinase 

LISÄTIETOA CD147:
Tämä välittää monilääkeresistenssiä syövässä.
 http://www.ncbi.nlm.nih.gov/pubmed/23623923

Havaittujen antigeenien ja reseptorien  hahmottamisesta kartta:
 http://www.med.unibs.it/~airc/sandra/fig_1-4.gif

MMP- kaskadi iskemisessä halvauksessa

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3615191/

Frontiers Media SA

Matrix Metalloproteinases and Blood-Brain Barrier Disruption in Acute Ischemic Stroke

Shaheen E. Lakhan, Annette Kirchgessner, [...], and Aidan Leonard

Additional article information

Abstract

Ischemic stroke continues to be one of the most challenging diseases in translational neurology. Tissue plasminogen activator (tPA) remains the only approved treatment for acute ischemic stroke, but its use is limited to the first hours after stroke onset due to an increased risk of hemorrhagic transformation over time resulting in enhanced brain injury. 

 In this review we discuss the role of matrix metalloproteinases (MMPs) in blood-brain barrier (BBB) disruption as a consequence of ischemic stroke.

 MMP-9 in particular appears to play an important role in tPA-associated hemorrhagic complications. Reactive oxygen species (ROS) can enhance the effects of tPA on MMP activation through the loss of caveolin-1 (cav-1), a protein encoded in the cav-1 gene that serves as a critical determinant of BBB permeability. 

 This review provides an overview of MMPs’ role in BBB breakdown during acute ischemic stroke. The possible role of MMPs in combination treatment of acute ischemic stroke is also examined.

Keywords: '

MMPs  matrix metalloproteinases, 

BBB blood-brain barrier,

 stroke, 

caveolin-1, 

ROS  reactive oxygen species

• Introduction

Stroke is the third leading cause of death in industrialized countries (Lo et al., 2003) and the most frequent cause of permanent disability in adults worldwide (Donnan et al., 2008). Acute ischemic stroke is the most common form of stroke and results from sudden blood vessel occlusion by a thrombus or embolism, resulting in an almost immediate loss of oxygen and glucose to the cerebral tissue. Although different mechanisms are involved in the pathogenesis of stroke, increasing evidence shows that ischemic injury and inflammation account for its pathogenic progression (Muir et al., 2007). Cerebral ischemia initiates cascades of pathological events, including vasogenic edema, disruption of the blood-brain barrier (BBB), intracranial hemorrhage (ICH), astroglial activation, and neuronal death. This ultimately causes irreversible neuronal injury in the ischemic core within minutes of the onset (Dimagl et al., 1999).

Despite advances in understanding the pathophysiology of cerebral ischemia, treatment options for acute ischemic stroke remain very limited (Donnan et al., 2008). Intravenous recombinant tissue plasminogen activator (tPA) remains the only FDA-approved thrombolytic therapy for reestablishing blood flow and salvaging brain tissue after acute ischemic stroke (Lijnen and Collen, 1987; National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, 1995). By degrading fibrin clots, tPA acts as a thrombolytic agent through the activation of plasminogen to plasmin (Lijnen and Collen, 1987).

Although tPA administered within 4.5h or less of symptom onset improves the functional outcome in patients (Miller et al., 2012; Wardlaw et al., 2012), it induces a 10-fold increase of symptomatic intracranial hemorrhage (ICH) (National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, 1995).

 Furthermore, delayed reperfusion with tPA beyond 3h is associated with an increased risk of hemorrhagic transformation (HT) with enhanced brain injury (Clark et al., 1999).

Moreover, tPA may cause injury to the BBB by activating matrix metalloproteinases (MMPs) (Wang et al., 2003).

 Thus, the therapeutic application of tPA is limited to specific clinical settings (National Institute of Neurological Disorders and Stroke t-PA Stroke Study Group, 1997). There is a pressing need to identify new combination therapies that can prevent tPA-associated ICH as well as extend the time window for thrombolysis without reducing its benefits.

Recent studies suggest that tPA adverse effects are mediated through MMPs, a family of >20 zinc-dependent enzymes that increase BBB permeability by degrading components of the extracellular matrix (ECM) and tight junctions (TJ) in endothelial cells (ECs) (Lapchak et al., 2000; Lijnen, 2001; Briasoulis et al., 2012).

 Increased expression and activation of MMPs plays a pivotal role in thrombolysis-mediated BBB leakage and edema, resulting in intracranial hemorrhage (Lapchak et al., 2000; Sumii and Lo, 2002). Reactive oxygen species (ROS) and its signaling pathways can enhance the effects of tPA on MMP activation (Harada et al., 2012).

•  In this review we provide an overview of the role of MMPs in BBB breakdown during acute ischemic stroke and the potential for MMP inhibition in the treatment of stroke.

• Structural Components of the BBB/Neurovascular Unit

The BBB is a dynamic interface between the peripheral circulation and the CNS. It controls the influx and efflux of biological substances needed for the brain metabolic processes, as well as for neuronal function. Thus, the functional and structural integrity of the BBB is vital in maintaining brain homeostasis.

The structure of the BBB has been discussed in reviews elsewhere (Sandoval and Witt, 2008; Abbott et al., 2010). Briefly, the anatomical substrate of the BBB is the cerebral microvascular endothelium, which together with the closely associated astrocytes, pericytes, neurons, and the ECM, constitute a “neurovascular unit” that is essential for the health and function of the CNS (del Zoppo, 2009). Cell–cell interactions in the neurovascular unit form the basis for brain function. Dysfunctional signaling in the neurovascular unit underlies the basis for disease. Alterations in microvessel integrity may have other effects within the neurovascular unit that affect neuronal function. The mechanisms of neurovascular unit response to stroke are not fully understood. However, any fully effective stroke therapy must include both prevention of cell death as well as repair of integrated neurovascular function.

The microcapillary endothelium is composed of TJs and follow a biphasic time course. Morphologically, BBB opening correlates with a redistribution of the TJ and AJ proteins from the plasma membrane to the cytoplasm as well as reorganization of the endothelial actin cytoskeleton.
 The extent of BBB disruption is associated with the type, severity, and duration of ischemic insults.
The molecular mechanisms underlying BBB opening are not fully understood, although several MMPs are believed to regulate BBB permeability and function during ischemic stroke (Mun-Bryce and Rosenberg, 1998).

The expression of MMPs in the adult brain is very low to undetectable, but clinical and experimental studies have shown that several MMPs are upregulated and activated after ischemic stroke (Lee et al., 2007; McColl et al., 2008). 

 MMPs disrupt the BBB by degrading the TJ proteins and basal lamina proteins, thereby leading to BBB leakage, leukocyte infiltration, brain edema, and hemorrhage. 

 Evidence suggests that MMP-2 and MMP-9 play different roles in BBB disruption during ischemic stroke.

•  MMP-2 KO( knock out)  does not provide neuroprotection in mouse models of permanent and transient MCAO (Asahi et al., 2001b). Consistently, in vitro data show that MMP-2 is not toxic to neurons in hippocampal slice preparations (Cunningham, 2005).

•  In contrast, MMP-9 KO (knock out)  provides strong neuroprotection in the same animal models, and in vitro MMP-9 is toxic to neurons in hippocampal slice preparations and in cultured primary cortical neurons (Asahi et al., 2000b).

In support of these data, a clinical study (Lucivero et al., 2007) reported an increase in plasma MMP-2 only in patients with lacunar (mild) stroke early (within 12h) and this was related to better outcome. In contrast, an increase in plasma MMP-9 was observed later (at day 7) and related to more severe stroke.

Matrix metalloproteinases are thought to have beneficial roles in stroke recovery. 

Shortly after an ischemic insult, a cascade of events is initiated in an attempt to repair the damage, a process similar to that found in wound healing (National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, 1995; Wardlaw et al., 2012). 

Following injury, blood vessels are dependent on the plasminogen activator system and

 on MMPs for their regeneration (Suzuki et al., 2009). 

It may be that a balanced level of MMP activity is important for vascular remodeling after ischemic brain injury (Yang and Rosenberg, 2011). Therefore, extended inhibition of MMPs, especially through the use of broad-spectrum inhibitors, might prove deleterious (National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group, 1995; Donnan et al., 2008).

• MMP-2 (gelatinase A)

Matrix metalloproteinase-2 is one of the two described human gelatinases in the MMP family, named for their ability to proteolytically degrade gelatine (denatured collagen) (see Table ​Table11 for a list of MMPs and their putative roles in acute ischemic stroke). MMP-2 is ubiquitously expressed as a 72-kDa proenzyme and subject to extensive glycosylation (Klein and Bischoff, 2011).

(KUVA)

• 3 tuntia halvauksesta   NNP-2:n aiheuttama  BBB läpivuoto

A  study provided indirect evidence that MMP-2 played a key role in initial opening of the BBB after cerebral ischemia (Rosenberg et al., 1998). In a rat model of transient MCAO, the initial opening of the BBB occurred as early as 3h after reperfusion and increased activation of MMP-2 correlated with the early opening of the BBB and the degradation of the TJ proteins claudin-5 and occludin in both cerebral hemispheres (Rosenberg et al., 1992; Yang et al., 2007). Experimental data clearly demonstrated an increase in MMP-2 at 3h, along with increased expression of the MMP-2 activators, MT1-MMP and furin.

A synthetic MMP inhibitor (BB-1101) blocked the increase in brain MMP-2 levels, but it did not have any effect on stroke lesion size at 48h after MCAO and had significant adverse effects on neurologic function in rats at 3 and 4weeks after MCAO (Rosenberg et al., 1992, 1998; Yang et al., 2007).
 In contrast, direct injection of MMP-2 into the rat brain resulted in the disruption of the BBB with subsequent hemorrhage, and this effect was inhibited by co-administration of TIMP-2 (Rosenberg et al., 1992). Thus, the early degradation of TJ proteins seems to be associated with a marked increase in MMP-2 in the early phase of ischemia.

Suofu et al. (2012) recently assessed the effects of MMP-2 KO, MMP-9 KO, and MMP-2/9 double KO (dKO) in protecting against mechanical reperfusion-induced HT and other brain injuries after the early stages of cerebral ischemia in mice of the same genetic background.

 Both MMP-2 and MMP-9 specifically attack the type IV collagen, laminin, and fibronectin, which are the major components of the basal lamina around the cerebral blood vessels. MCAO was performed and reperfusion was started at 1 or 1.5h after onset of MCAO. Mice were sacrificed 8h later. Both pro- and active-MMP-2 and MMP-9 levels were significantly elevated in the early ischemic brain. After the early stages of ischemia and reperfusion, the hemorrhagic incidence was reduced in the cortex of MMP-2 KO mice. The hemorrhagic volume was also significantly decreased in the cortexes of MMP-2 and/or -9 KO mice. In the basal ganglia, MMP-2 KO and MMP-2/9 dKO mice displayed a remarkable decrease in hemorrhagic volume, but MMP-9 deletion did not protect against hemorrhage. MMP-2 and/or -9 KO mice displayed significantly decreased infarction volume in both the cortex and striatum, in addition to improved neurological function. The results suggested that MMP-2 deficiency as well as MMP-2 and MMP-9 double deficiency were more protective than MMP-9 deficiency alone against HT after the early stages of ischemia and reperfusion.

• MMP-3 (stromelysin-1)

Matrix metalloproteinase-3 (stromelysin-1) was first described in 1985 as a 51-kDa protein secreted by rabbit fibroblasts (Chin et al., 1985). MMP-3 could be distinguished from collagenases by the inability to degrade type I collagen. The substrate specificity of MMP-3 is broad and MMP-3 has been found to degrade

JATKUU (Suomennettava myöh,)

Mitä tPA vaikuttaa MMP- kaskadiin? Yleistä tPA/plasminogeeni-akselista.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1383744/

Thrombosis and haemostasis

Author Manuscript

NIH Public Access

Tissue plasminogen activator in central nervous system physiology and pathology

Jerry P. Melchor and Sidney Strickland

Additional article information

Summary

Although conventionally associated with fibrin clot degradation, recent work has uncovered new functions for the tissue plasminogen activator (tPA)/plasminogen cascade in central nervous system physiology and pathology. This extracellular proteolytic cascade has been shown to have roles in learning and memory, stress, neuronal degeneration, addiction and Alzheimer’s disease. The current review considers the different ways tPA functions in the brain.

Lähdeartikkelin lyhennyksiä

PA: plasminogen activator,

 tPA: tissue-type plasminogen activator,

 uPA: urokinase-type plasminogen activator, 

PAI-1: plasminogen activator inhibitor-1, 

LRP: low-density lipoprotein receptor-related protein, (tPA clearing receptor)

 CNS: central nervous system, 

CPEB: cytoplasmic polyadenylation element binding,

LTP: long-term potentiation, 

LTD: long-term depression,

 mRNA: messenger ribonucleic acid,

 BDNF: brain-derived neurotrophic factor,

 NMDA: N-methyl-D-aspartate,

 RAP: receptor-associated protein,

 ECM: extracellular matrix, 

BBB: blood brain barrier, 

MMP: matrix metalloproteinase, 

EtOH: ethanol,

 ERK: extracellular signal-regulated kinase, 

GAP: growth-associated protein, 

CRF: corticotropin-releasing factor,

MCAO: middle cerebral artery occlusion, 

Aβ: beta-amyloid

Sitaatti artikkelista:  

Johdannosta (Introduction)

 The plasminogen activator (PA)/plasminogen proteolytic cascade is known to be important for thrombolysis (1, 2). There are two mammalian PAs: tissue-type (tPA) and urokinase-type (uPA) (3).
 PAs are serine proteases that cleave a specific peptide bond within the zymogen plasminogen to generate the active protease, plasmin, which is capable of degrading numerous substrates
In the vasculature, plasmin efficiently breaks down fibrin clots, aiding in hemostasis and vascular patency. This protease cascade is tightly regulated by the actions of serine protease inhibitors (serpins), of which plasminogen activator inhibitor-1 (PAI-1) and neuroserpin are the major cognate serpins for tPA, while plasmin is inhibited by α₂ -antiplasmin (2).
 Furthermore, tPA activity can be attenuated by rapid clearance through low-density lipoprotein receptor-related protein (LRP)-mediated endocytosis and augmented by binding annexin-II or to fibrin within a clot (4, 5).

Infusion of PAs, such as recombinant tPA and its derivatives, is used for lysis of fibrin clots to help restore blood flow following myocardial infarction or thrombotic stroke (2). Although tPA can be used for the treatment of stroke, there is a narrow time-frame and limited patient population for which thrombolytics are appropriate, since delayed delivery of tPA can lead to neuronal damage or cerebral hemorrhage, worsening the outcome for the patient.

The function of tPA, however, is not limited to the initiation of thrombolysis. Recent research has shown that the tPA/plasminogen system has other roles within the central nervous system (CNS). Although the presence of uPA has been confirmed in the brain, its role has not been fully investigated. Therefore, this review will focus on the various functions of tPA in brain physiology and pathology.

• tPA/plasminogen system in CNS physiology

tPA expression and regulation

tPA is highly expressed in the adult mouse brain in regions involved in learning and memory (hippocampus), fear and anxiety (amygdala), motor learning (cerebellum), and autonomic and endocrine functions (hypothalamus) (6–13). Both neurons and microglial cells express tPA.
Plasminogen, PAI-1, and neuroserpin are also present in the brain indicating that the components of the tPA/plasminogen cascade are present in the CNS (9, 13–15).

 In neurons, the expression of tPA can be under translational control; tPA expression is regulated by the binding of the cytoplasmic polyadenylation element binding (CPEB) protein which leads to the extension of tPA mRNA polyadenylation and a subsequent increase in tPA protein synthesis (16). This regulation of tPA expression allows for a rapid increase in activity in response to specific stimuli (as seen in synaptic plasticity), and the presence of cognate serpins of tPA in the CNS allows for swift and specific inactivation. The localization to axon terminals and the highly regulated axonal release of tPA is consistent with a proteolytic-dependent role for tPA in the CNS in which PA cascade activation alters the extracellular matrix (ECM) and modifies synapses, such as during seizure, kindling, and long-term potentiation (LTP) (6, 17).

While tPA also has been visualized in dendrites, the release of this protein from these structures has yet to be demonstrated (16).

There is an additional level of complexity to tPA expression in neuronal cells. In neurons, tPA is not constitutively secreted but contained in vesicles. tPA can be released from secretory vesicles after membrane depolarization or stimulation (18–21). This mechanism of regulated tPA secretion allows for rapid localized increase in tPA activity at the synapse.

• The role of tPA in synaptic plasticity

Long-term potentiation (LTP), considered to be a molecular correlate of learning, refers to remodeling of neuronal connections leading to increased synaptic strength after repetitive excitatory stimulation (22). tPA was identified as an immediate-early gene whose mRNA transcription is induced shortly after synaptic activity such as LTP (6). The possibility that this protease participates in synaptic plasticity is strengthened by the fact that tPA is elevated after LTP. Indeed, either inhibition of proteolytic activity or deficiency in tPA affects the late-phase of LTP (L-LTP) or long-term depression (LTD), while increased tPA facilitates LTP and learning (21, 23).

 Another action of the tPA/plasminogen system that contributes to LTP generation is cleavage of the pre-cursor form of brain-derived neurotrophic factor (proBDNF) to its mature form (mBDNF) (24). The application of mBDNF, derived from tPA/plasmin cleavage, to hippocampal slices can rescue impaired L-LTP in both tPA-deficient and plasminogen-deficient mice. This work reveals an interaction between the tPA/plasminogen axis and BDNF and suggests a potential impact of this interaction on LTP. These observations suggest further studies to define plasmin/proBDNF binding and identify putative mBDNF receptors for LTP propagation.

One of the molecules with which tPA purportedly interacts to enhance LTP is the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor. Using a neuronal cell culture model, Nicole et al. reported that tPA cleaves NR1, enhancing NMDA-mediated intracellular calcium levels and neuronal degeneration (25). In this model, tPA cleaves at amino acid Arg260 of the NR1 subunit leading to NMDA signaling enhancement; mutation of Arg260 to Ala260 abrogates tPA-induced NMDA activity augmentation (26). However, the direct cleavage of NR1 by tPA has not been observed in other studies (27, 28). An interaction between tPA and NR2B-containing NMDA receptors has been reported during ethanol withdrawal-induced seizures (29). Therefore, although tPA can interact with either NR1 or NR2B, the specific outcome of these interactions as they pertain to LTP have yet to be delineated.

An additional tPA receptor affecting synaptic plasticity is LRP (LDL-receptor related protein)  An antagonist of LRP, receptor-associated protein (RAP), hinders tPA-induced L-LTP and prevents rescue of synaptic potentiation by addition of tPA in tPA-deficient mice (17).

 Additionally, the binding of tPA to LRP can lead to the up-regulation of matrix metalloproteinase-9 (MMP-9), a protease which can degrade the ECM contributing to either synaptic plasticity or neuronal degeneration (30). 

Interaction between LRP and tPA also contributes to blood-brain barrier (BBB) breakdown (31). tPA-induced opening of the BBB is evident in both plasminogen-deficient and MMP-9-deficient mice as measured by Evans blue dye extravasation, implying that neither plasminogen nor MMP-9 is necessary for tPA to produce its effect on vascular permeability.

 However, the tPA-clearing receptor LRP is involved since both RAP and anti-LRP antibodies can block this specific tPA activity. The exact mechanism by which tPA alters the BBB is still not clear and is subject to further investigation.

The function of tPA in plasticity in vivo has been scrutinized in mice either deficient for or over-expressing this protease since Qian and co-workers showed that tPA mRNA was up-regulated in the rat hippocampus after seizure, kindling, and LTP (6). Although several studies do not indicate a role for tPA in traditional hippocampal-based behavioral tests, mice overexpressing tPA in the CNS show enhanced LTP in hippocampal slices and exhibit improved hippocampal-dependent spatial memory formation as measured by Morris water maze and homing hole board tests (32). In these studies, overexpression of tPA led to increased synaptic activity in vivo, which is similar to published in vitro results. Thus, despite a lack of a learning phenotype in tPA-deficient mice in several learning paradigms, behavioral results from tPA-overexpressing mice indicate a role for this protease in memory (23, 33).

tPA has also been studied in the amygdala, a brain region that regulates reponse to fear and anxiety (10, 12). Acute restraint stress studies in mice indicate that one function of this protease is in amygdala-dependent learning in fear response. tPA is induced in the medial and central amygdala after acute restraint stress, where it promotes stress-induced post-synaptic (phosphorylation of ERK1/2) and axonal (amplified GAP-43 expression) neuronal changes (10). tPA-deficient mice display impaired response to stress and abnormal circulating levels of corticosterone during the recovery period after stress. They also do not exhibit stress-induced anxiety as measured by the elevated-plus maze test. These results indicate that tPA contributes to proper control of hormonal stress response and has a significant role in emotional learning. 

Further research into the role of tPA in stress has revealed that this protease is elevated after the infusion of corticotrophin-releasing factor (CRF), an important hormone for triggering the stress response, into the lateral ventricle (34). The activation of tPA by CRF in the central and medial amygdala leads to an increase of c-fos immunoreactivity, a measure of neuronal activation. However, the function of tPA seems to be independent of plasmin production during stress-induced anxiety, since plasminogen-deficient mice, unlike tPA-deficient mice, do not show lower levels of anxiety in the elevated plus-maze after restraint stress or abnormal c-fos expression after CRF infusion into the ventricle. As the function of tPA in stress and the amygdala have been uncovered, the next challenge is to identify new therapies for anxiety-related disorders in the context of the tPA/plasminogen axis.

Another area of the brain in which tPA has been shown to have a role is the cerebellum, which is responsible for motor learning. Seeds and colleagues reported the up-regulation of tPA mRNA in rats after learning a complicated motor task (traversing a runway by grabbing horizontal irregular pegs) (7). Additionally, even though there were no apparent consequences, the rate of cerebellar granule cell migration seems hindered in tPA-deficient mice, since more granule cells are present in the molecular layer of the cerebellum in these mice as compared to age-matched controls (8). Consistent with the importance of proteolytic action, wild-type mice infused with tPA inhibitors PAI-1 or tPA-STOP have deficits in cerebellar motor learning (11).

In addition to its roles in the hippocampus, amygdala, and cerebellum, the tPA/plasminogen system is also involved in the recovery of function in the visual cortex after reverse occlusion. The development of the visual pathway is dependent on activity and leads to ocular dominance, a condition in which certain cortical neurons become selectively connected to one specific eye. Ocular dominance progression can be enhanced by addition of norepinephrine, which leads to increased tPA mRNA (35). Preventing visual stimulation of an eye leads to monocular deprivation, a state of diminished cortical response in the closed eye. Reverse occlusion is the rescue of visual function in the previously deprived eye by opening this eye and closing the formerly open eye, and this process occurs by the formation of connections from the lateral geniculate nucleus of the thalamus to the visual cortex.
 Muller and Griesinger have shown the tPA/plasminogen proteolytic cascade is necessary for reverse occlusion, since inhibition of either protease prevents proper formation of thalamocortical connections for visual rescue (36). Other research has extended this finding, indicating that tPA/plasminogen activity allows for reorganization of connections in the visual cortex, again highlighting this proteolytic cascade’s contribution in synaptic plasticity (37). More specifically, two recent reports show that during monocular deprivation, tPA/plasminogen activity helps in structural remodeling by pruning dendritic spines and reorganizing the ECM (38, 39).

Addiction can be considered a form of adaptive synaptic plasticity. In models of morphine and ethanol addiction, tPA expression is elevated in the nucleus accumbens and limbic system, respectively. The rewarding effects of morphine can be measured by the conditioned place-preference test, which associates the rewarding or aversive effect of a drug to placement into a specific compartment. Both the rewarding effect of morphine and morphine- related dopamine release are diminished in tPA-deficient and plasminogen-deficient mice when compared to wild-type mice (40). Although the effector substrate of plasmin in morphine addiction is not yet identified, the role of plasmin in this pathway is evident.

In addition to morphine addiction, ethanol consumption and withdrawal also elevate tPA activity. Ethanol inhibits NMDA receptor activity, and NMDA receptor numbers increase as an adaptive response. The rapid removal of ethanol relieves this inhibition on the expanded population of NMDA receptors, and when tPA acts on this large number of NR2B-containing NMDA receptors, it can result in hyperexcitation and seizures. However, in this instance, the primary effect of tPA does not appear to be plasminogen activation but rather interaction with the NR2B subunit of the NMDA receptor in a non-proteolytic manner (29). Additionally, tPA-deficient mice also have reduced ethanol withdrawal seizures (see below). This is an example of the progression of a physiological role for tPA (synaptic plasticity in addiction) resulting in a pathological consequence (seizure induction upon ethanol withdrawal).

• tPA function in CNS pathology

The role of tPA in neurotoxicity

While the understanding of physiological functions of tPA in the CNS has expanded, so has understanding of its roles in pathological situations. The participation of the tPA/plasminogen axis has been defined in neuronal degeneration due to excitotoxicity. Injection of kainate, an excitotoxic glutamate analog, into the CA1 region of the hippocampus of wild-type mice leads to neuronal damage, and this damage is reduced in either tPA-deficient or plasminogen-deficient mice (41–43). Neurodegeneration is prevented in wild-type mice co-injected with both kainate and α₂ -antiplasmin, supporting the role of plasmin in this neuronal injury paradigm.

Further investigation identified laminin, a component of the ECM, as a substrate of plasmin proteolysis, and laminin cleavage combined with glutamate excitotoxicity results in neuronal degeneration due to anoikis (43–46). Plasmin-cleaved laminin fragments or anti-laminin antibodies infused into the hippocampus of either wild-type or plasminogen-deficient mice disrupts the ECM, sensitizing these mice to kainate excitotoxicity (46). The disruption of the ECM by laminin fragments indicates that the ECM is an active structure, whose components can be replaced or competed against by excess exogenous laminin. The dynamic nature of the ECM would also permit the remodeling necessary during synaptic plasticity, and it has been reported that cleavage of laminin by plasmin can hinder LTP in rat hippocampal neurons (47).

While the tPA/plasminogen axis contributes to excitotoxic neuronal degeneration, tPA is also involved in other CNS pathologies. The role of tPA has been studied in Alzheimer’s disease (AD), stroke, infarct formation, and seizure spreading. tPA expression is elevated during seizures, a state of synchronous, pathological hyperactivity in the CNS. Comparable to observations in the hippocampus, delivery of kainate into the rat amygdala results in damage to hippocampal neurons (48). The neurodegeneration observed in the hippocampus after kainate injection into the amygdala is tPA-dependent, since neuronal damage can be attenuated by neuroserpin delivery into the brain (48). Additionally, seizure spreading is plasminogen-independent, since seizure inception in plasminogen-deficient mice was similar to wild-type mice, and seizure onset is also delayed by neuroserpin. The tPA/plasminogen system also contributes to handling-induced seizures after ethanol withdrawal in mice, and these seizures are attenuated in tPA-deficient mice (29).

The role of tPA in infarct formation has also been described. tPA-deficient mice have reduced infarct volume and neuronal damage as compared to control mice subjected to middle cerebral artery occlusion (MCAO) (49, 50). The infusion of recombinant tPA or delivery of tPA-expressing adenovirus into tPA-deficient mice also led to increased infarct size after MCAO, indicating a role for tPA in this process. The observation that thrombolytics can increase the infarct size after MCAO is independent of plasminogen activation since plasminogen-deficient mice displayed larger infarcts post-MCAO. In accordance with the contribution of tPA to infarct formation, PAI-1-deficient mice have increased infarct size while neuroserpin-injected mice have diminished infarcts after MCAO. Therefore, regulating tPA activity might be beneficial in controlling infarct injury after stroke (50–52).

• tPA in Alzheimer’s disease

AD is the most common cause of dementia and cognitive decline in the elderly (53). One of the characteristic pathologies of AD is the deposition of a 39–43 amino acid peptide called β-amyloid (Aβ) in the CNS parenchyma, leading to the activation of the inflammatory response. The PAI-1 gene is induced during inflammation, which results in depressed tPA activity in transgenic AD mouse models (54–56). Furthermore, the tPA/plasminogen system has been implicated in the degradation of A β in the parenchyma of both mice and humans (56–60). Diminished tPA/plasmin activity in the AD brain can contribute to a deleterious cycle in which increased A β concentration leads to elevation of PAI-1, further depressing tPA/plasmin activity and resulting in inefficient A β degradation. Concomitant with inflammation-induced increase in PAI-1 levels, decreased plasmin activity in AD brains may also be due to diminished plasminogen availability in lipid rafts, areas in neurons where plasminogen and plasminogen-binding molecules reside (61). These rafts are disturbed in AD brains, consequently resulting in decreased plasmin activity. These observations indicate that molecules within the tPA/plasminogen proteolytic axis are viable therapeutic targets to delay the progression of AD.

• Serpins in CNS pathology

There is a significant elevation in PAI-1 and neuroserpin expression during pathological insult to the brain. The increase of PAI-1 in the brain is seen in neurological disorders such as AD and dementia, after kainate injection into the CA1 region of the hippocampus, and after restraint stress. Escalation of PAI-1 expression normally corresponds to a depression of tPA activity, and it is thought to be a regulatory response to increased tPA, since excessive tPA activity can contribute to CNS pathology, such as by worsening neuronal damage. However, PAI-1 could interact with tPA, not as a serpin but as a binding partner to mediate its effect on receptors including the NMDA receptors. Clearly, tPA can function either as a protease or modulator of cell signaling, and PAI-1 can influence both of these activities. The elevation of neuroserpin, the other major tPA inhibitor in the CNS, has also been implicated in regulation of seizure spreading, control of BBB integrity, and emotional learning (15, 62, 63).

• Conclusions

The function of the tPA/plasminogen proteolytic cascade has been conventionally assigned to fibrinolysis. However, existing research, especially using mice deficient in specific components of the fibrinolytic system, has extended the role of tPA and plasminogen from thrombolytic enzymes to encompass functions in both CNS physiology and pathology (Table I). Identification of novel substrates for either tPA or plasmin in the CNS would extend understanding of the influence of this proteolytic cascade. New lessons learned about the function of the tPA/plasminogen system can be utilized to identify novel therapies for slowing the pathogenesis of disorders such as AD or attenuating the severity of ethanol withdrawal-induced seizures. Further clarification of the contributions of this proteolytic cascade to LTP (such as the identification of the interaction domains between tPA and NR2B or proBDNF and plasmin) might help ease cognitive decline. The potential applications of new functions of the tPA/plasminogen proteolytic cascade highlight the importance and complexity of this system.