Characterization of metabolic alterations in the lean metabolically unhealthy alpha defensin transgenic mice.

Inflammation is consistently linked to dysmetabolism. In transgenic mice (Def+/+) model the neutrophilic peptide, alpha defensin, proved atherogenic. This phenotype occurred despite favorable cholesterol and glucose levels, and lower body weight and blood pressure. In this study, integration of metabolic&behavioral phenotyping system, endocrine, biochemical and mitochondrial assessment, pathological and immunohistochemical tests, and multiple challenge tests was established to explore the metabolic impact of alpha defensin. Compared to the control group, Def+/+ mice exhibited lower total energy expenditure and carbohydrate utilization, and higher fat oxidation. Their ACTH-cortisol and thyroid profiles were intact. Intriguingly, they had low levels of glucagon, with high ammonia, uric acid, triglyceride, and lactate. Mitochondrial evaluations were normal. Overall, defensin-induced hypoglucagonemia is associated with lipolysis, restricted glucose oxidation, and enhanced wasting. Def+/+ mice may be a useful model for studying the category of lean, apparently metabolically healthy, and atherosclerotic phenotype, with insight into a potential inflammatory-metabolic link.

Alleviation of Hepatic Steatosis by Alpha-Defensin Is Associated with Enhanced Lipolysis.

Background and Objectives: The neutrophilic peptide, alpha-defensin, is considered an evolving risk factor intimately linked with lipid mobilization. It was previously linked to augmented liver fibrosis. Here, we assess a potential association between alpha-defensin and fatty liver. Materials and Methods: A cohort of transgenic C57BL/6JDef+/+ male mice that overexpress the human neutrophil-derived alpha-defensin in their polymorphonuclear neutrophils (PMNs) were assessed for liver steatosis and fibrosis development. Wild type (C57BL/6JDef.Wt) and transgenic (C57BL/6JDef+/+) mice were maintained on a standard rodent chow diet for 8.5 months. At the termination of the experiment, systemic metabolic indices and hepatic immunological cell profiling were assessed. Results: The Def+/+ transgenic mice exhibited lower body and liver weights, lower serum fasting glucose and cholesterol, and significantly lower liver fat content. These results were associated with impaired liver lymphocytes count and function (lower CD8, NK cells, and killing marker CD107a). The metabolic cage demonstrated dominant fat utilization with a comparable food intake in the Def+/+ mice. Conclusions: Chronic physiological expression of alpha-defensin induces favorable blood metabolic profile, increased systemic lipolysis, and decreased hepatic fat accumulation. Further studies are needed to characterize the defensin net liver effect.

Divergent impacts of tocilizumab and colchicine in COVID-19-associated coagulopathy: the role of alpha-defensins.

Patients who are severely affected by coronavirus disease 2019 (COVID-19) may develop a delayed onset 'cytokine storm', which includes an increase in interleukin-6 (IL-6). This may be followed by a pro-thrombotic state and increased D-dimers. It was anticipated that tocilizumab (TCZ), an anti-IL-6 receptor monoclonal antibody, would mitigate inflammation and coagulation in patients with COVID-19. However, clinical trials with TCZ have recorded an increase in D-dimer levels. In contrast to TCZ, colchicine reduced D-dimer levels in patients with COVID-19. To understand how the two anti-inflammatory agents have diverse effects on D-dimer levels, we present data from two clinical trials that we performed. In the first trial, TCZ was administered (8 mg/kg) to patients who had a positive polymerase chain reaction test for COVID-19. In the second trial, colchicine was given (0·5 mg twice a day). We found that TCZ significantly increased IL-6, α-Defensin (α-Def), a pro-thrombotic peptide, and D-dimers. In contrast, treatment with colchicine reduced α-Def and Di-dimer levels. In vitro studies show that IL-6 stimulated the release of α-Def from human neutrophils but in contrast to colchicine, TCZ did not inhibit the stimulatory effect of IL-6; raising the possibility that the increase in IL-6 in patients with COVID-19 treated with TCZ triggers the release of α-Def, which promotes pro-thrombotic events reflected in an increase in D-dimer levels.

Alpha-defensins: risk factor for thrombosis in COVID-19 infection.

The inflammatory response to SARS/CoV-2 (COVID-19) infection may contribute to the risk of thromboembolic complications. α-Defensins, antimicrobial peptides released from activated neutrophils, are anti-fibrinolytic and prothrombotic in vitro and in mouse models. In this prospective study of 176 patients with COVID-19 infection, we found that plasma levels of α-defensins were elevated, tracked with disease progression/mortality or resolution and with plasma levels of interleukin-6 (IL-6) and D-dimers. Immunohistochemistry revealed intense deposition of α-defensins in lung vasculature and thrombi. IL-6 stimulated the release of α-defensins from neutrophils, thereby accelerating coagulation and inhibiting fibrinolysis in human blood, imitating the coagulation pattern in COVID-19 patients. The procoagulant effect of IL-6 was inhibited by colchicine, which blocks neutrophil degranulation. These studies describe a link between inflammation and the risk of thromboembolism, and they identify a potential new approach to mitigate this risk in patients with COVID-19 and potentially in other inflammatory prothrombotic conditions.

ACE2, COVID-19 Infection, Inflammation, and Coagulopathy: Missing Pieces in the Puzzle.

Engulfed by the grave consequences of the coronavirus disease 2019 (COVID-19) pandemic, a better understanding of the unique pattern of viral invasion and virulence is of utmost importance. Angiotensin (Ang)-converting enzyme (ACE) 2 is a key component in COVID-19 infection. Expressed on cell membranes in target pulmonary and intestinal host cells, ACE2 serves as an anchor for initial viral homing, binding to COVID-19 spike-protein domains to enable viral entry into cells and subsequent replication. Viral attachment is facilitated by a multiplicity of membranal and circulating proteases that further uncover attachment loci. Inherent or acquired enhancement of membrane ACE2 expression, likely leads to a higher degree of infection and may explain the predisposition to severe disease among males, diabetics, or patients with respiratory or cardiac diseases. Additionally, once attached, viral intracellular translocation and replication leads to depletion of membranal ACE2 through degradation and shedding. ACE2 generates Ang 1-7, which serves a critical role in counterbalancing the vasoconstrictive, pro-inflammatory, and pro-coagulant effects of ACE-induced Ang II. Therefore, Ang 1-7 may decline in tissues infected by COVID-19, leading to unopposed deleterious outcomes of Ang II. This likely leads to microcirculatory derangement with endothelial damage, profound inflammation, and coagulopathy that characterize the more severe clinical manifestations of COVID-19 infection. Our understanding of COVID-ACE2 associations is incomplete, and some conceptual formulations are currently speculative, leading to controversies over issues such as the usage of ACE inhibitors or Ang-receptor blockers (ARBs). This highlights the importance of focusing on ACE2 physiology in the evaluation and management of COVID-19 disease.

Opposing effects of HNP1 (α-defensin-1) on plasma cholesterol and atherogenesis.

Atherosclerosis, the predominant cause of death in well-resourced countries, may develop in the presence of plasma lipid levels within the normal range. Inflammation may contribute to lesion development in these individuals, but the underlying mechanisms are not well understood. Transgenic mice expressing α-def-1 released from activated neutrophils develop larger lipid and macrophage-rich lesions in the proximal aortae notwithstanding hypocholesterolemia caused by accelerated clearance of α-def-1/low-density lipoprotein (LDL) complexes from the plasma. The phenotype does not develop when the release of α-def-1 is prevented with colchicine. However, ApoE-/- mice crossed with α-def-1 mice or given exogenous α-def-1 develop smaller aortic lesions associated with reduced plasma cholesterol, suggesting a protective effect of accelerated LDL clearance. Experiments were performed to address this seeming paradox and to determine if α-def-1 might provide a means to lower cholesterol and thereby attenuate atherogenesis. We confirmed that exposing ApoE-/- mice to α-def-1 lowers total plasma cholesterol and decreases lesion size. However, lesion size was larger than in mice with total plasma cholesterol lowered to the same extent by inhibiting its adsorption or by ingesting a low-fat diet. Furthermore, α-def-1 levels correlated independently with lesion size in ApoE-/- mice. These studies show that α-def-1 has competing effects on atherogenesis. Although α-def-1 accelerates LDL clearance from plasma, it also stimulates deposition and retention of LDL in the vasculature, which may contribute to development of atherosclerosis in individuals with normal or even low plasma levels of cholesterol. Inhibiting α-def-1 may attenuate the impact of chronic inflammation on atherosclerotic vascular disease.

Release of IL-6 After Stroke Contributes to Impaired Cerebral Autoregulation and Hippocampal Neuronal Necrosis Through NMDA Receptor Activation and Upregulation of ET-1 and JNK.

The sole FDA-approved drug treatment for ischemic stroke is tissue-type plasminogen activator (tPA). However, upregulation of JNK mitogen-activated protein kinase (MAPK) and endothelin 1 (ET-1) by tPA after stroke contributes to impaired cerebrovascular autoregulation. Wild-type (wt) tPA can bind to the lipoprotein-related receptor (LRP), which mediates vasodilation, or NMDA receptors (NMDA-Rs), exacerbating vasoconstriction. Elevations in IL-6, a marker of inflammation that accompanies stroke, are reported to be an adverse prognostic factor. We hypothesized that IL-6 released into CSF after stroke by wt-tPA through activation of NMDA-Rs and upregulation of ET-1 and JNK contribute to impairment of cerebrovascular autoregulation and increased histopathology. Results show that IL-6 was increased post stroke in pigs, which was increased further by wt-tPA. Co-administration of the IL-6 antagonist LMT-28 with wt-tPA prevented impairment of cerebrovascular autoregulation and necrosis of hippocampal cells. wt-tPA co-administered with the JNK inhibitor SP 600125 and the ET-1 antagonist BQ 123 blocked stroke-induced elevation of IL-6. Co-administration of LMT-28 with wt-tPA blocked the augmentation of JNK and ET-1 post stroke. In conclusion, IL-6 released after stroke, which is enhanced by wt-tPA through activation of NMDA-Rs and upregulation of ET-1 and JNK, impairs cerebrovascular autoregulation and increases histopathology. Strategies that promote fibrinolysis while limiting activation of NMDA-Rs and upregulation of IL-6 may improve the benefit/risk ratio compared to wt-tPA in treatment of stroke.

Neutrophil α-defensins promote thrombosis in vivo by altering fibrin formation, structure, and stability.

Inflammation and thrombosis are integrated, mutually reinforcing processes, but the interregulatory mechanisms are incompletely defined. Here, we examined the contribution of α-defensins (α-defs), antimicrobial proteins released from activated human neutrophils, on clot formation in vitro and in vivo. Activation of the intrinsic pathway of coagulation stimulates release of α-defs from neutrophils. α-Defs accelerate fibrin polymerization, increase fiber density and branching, incorporate into nascent fibrin clots, and impede fibrinolysis in vitro. Transgenic mice (Def++) expressing human α-Def-1 developed larger, occlusive, neutrophil-rich clots after partial inferior vena cava (IVC) ligation than those that formed in wild-type (WT) mice. IVC thrombi extracted from Def++ mice were composed of a fibrin meshwork that was denser and contained a higher proportion of tightly packed compressed polyhedral erythrocytes than those that developed in WT mice. Def++ mice were resistant to thromboprophylaxis with heparin. Inhibiting activation of the intrinsic pathway of coagulation, bone marrow transplantation from WT mice or provision of colchicine to Def++ mice to inhibit neutrophil degranulation decreased plasma levels of α-defs, caused a phenotypic reversion characterized by smaller thrombi comparable to those formed in WT mice, and restored responsiveness to heparin. These data identify α-defs as a potentially important and tractable link between innate immunity and thrombosis.

tPA variant tPA-A296-299 Prevents impairment of cerebral autoregulation and necrosis of hippocampal neurons after stroke by inhibiting upregulation of ET-1.

Tissue-type plasminogen activator (tPA) is neurotoxic and exacerbates uncoupling of cerebral blood flow (CBF) and metabolism after stroke, yet it remains the sole FDA-approved drug for treatment of ischemic stroke. Upregulation of c-Jun-terminal kinase (JNK) after stroke contributes to tPA-mediated impairment of autoregulation, but the role of endothelin-1 (ET-1) is unknown. Based on the Glasgow Coma Scale, impaired autoregulation is linked to adverse outcomes after TBI, but correlation with hippocampal histopathology after stroke has not been established. We propose that given after stroke, tPA activates N-Methyl-D-Aspartate receptors (NMDA-Rs) and upregulates ET-1 in a JNK dependent manner, imparing autoregulation and leading to histopathology. After stroke, CBF was reduced in the hippocampus and reduced further during hypotension, which did not occur in hypotensive sham pigs, indicating impairment of autoregulation. Autoregulation and necrosis of hippocampal CA1 and CA3 neurons were further impaired by tPA, but were preserved by the ET-1 antagonist BQ 123 and tPA-A,296-299 a variant that is fibrinolytic but does not bind to NMDA-Rs. Expression of ET-1 was increased by stroke and potentiated by tPA but returned to sham levels by tPA-A296-299 and the JNK antagonist SP600125. Results show that JNK releases ET-1 after stroke. Tissue-type plasminogen activator -A296-299 prevents impairment of cerebral autoregulation and histopathology after stroke by inhibiting upregulation of ET-1.

Tissue-Type Plasminogen Activator-A296-299 Prevents Impairment of Cerebral Autoregulation After Stroke Through Lipoprotein-Related Receptor-Dependent Increase in cAMP and p38.

BACKGROUND AND PURPOSE: The sole Food and Drug Administration-approved treatment for stroke is tissue-type plasminogen activator (tPA), but its brief therapeutic window and complications of treatment constrain its use. One limitation may be its potential to exacerbate impairment of cerebral autoregulation after stroke. Vasodilation is maintained by elevations in cAMP. However, cAMP levels fall after stroke because of overactivation of N-methyl-d-aspartate receptors by toxic levels of glutamate, an effect that is exacerbated by tPA. Binding of wild-type (wt) tPA to the low-density lipoprotein-related receptor (LRP) mediates dilation. We propose that binding of wt-tPA to N-methyl-d-aspartate receptor reduces cAMP and impairs vasodilation. We hypothesize that tPA-A(296-299), a variant that is fibrinolytic but cannot bind to N-methyl-d-aspartate receptor, preferentially binds to LRP and increases cAMP and p38, limiting autoregulation impairment after stroke. METHODS: Stroke was induced by photothrombosis in pigs equipped with a closed cranial window, cerebral blood flow determined by microspheres, and cerebrospinal fluid cAMP and p38 determined by ELISA. RESULTS: Stroke decreased cerebral blood flow. Cerebral blood flow was reduced further during hypotension, indicating impairment of autoregulation. Autoregulation was further impaired by wt-tPA, which was prevented by MK801 and tPA-A(296-299). Protection by tPA-A(296-299) was blocked by anti-LRP Ab, the LRP antagonist receptor-associated protein, and the p38 inhibitor SB 203580, but not by control IgG. Stroke reduced cerebrospinal fluid cAMP, which was reduced further by wt-tPA, but augmented by tPA-A(296-299). Cerebrospinal fluid p38 was unchanged by wt-tPA, increased by tPA-A(296-299), and decreased by anti-LRP Ab and receptor-associated protein. CONCLUSIONS: tPA-A(296-299) prevents impairment of cerebral autoregulation after stroke through an LRP-dependent increase in cAMP and p38.

α-Defensins Induce a Post-translational Modification of Low Density Lipoprotein (LDL) That Promotes Atherosclerosis at Normal Levels of Plasma Cholesterol.

Approximately one-half of the patients who develop clinical atherosclerosis have normal or only modest elevations in plasma lipids, indicating that additional mechanisms contribute to pathogenesis. In view of increasing evidence that inflammation contributes to atherogenesis, we studied the effect of human neutrophil α-defensins on low density lipoprotein (LDL) trafficking, metabolism, vascular deposition, and atherogenesis using transgenic mice expressing human α-defensins in their polymorphonuclear leukocytes (Def(+/+)). Accelerated Def(+/+) mice developed α-defensin·LDL complexes that accelerate the clearance of LDL from the circulation accompanied by enhanced vascular deposition and retention of LDL, induction of endothelial cathepsins, increased endothelial permeability to LDL, and the development of lipid streaks in the aortic roots when fed a regular diet and at normal plasma levels of LDL. Transplantation of bone marrow from Def(+/+) to WT mice increased LDL clearance, increased vascular permeability, and increased vascular deposition of LDL, whereas transplantation of WT bone marrow to Def(+/+) mice prevented these outcomes. The same outcome was obtained by treating Def(+/+) mice with colchicine to inhibit the release of α-defensins. These studies identify a potential new link between inflammation and the development of atherosclerosis.

Endogenous plasminogen activators mediate progressive intracerebral hemorrhage after traumatic brain injury in mice.

Persistent intracerebral hemorrhage (ICH) is a major cause of death and disability after traumatic brain injury (TBI) for which no medical treatment is available. Delayed bleeding is often ascribed to consumptive coagulopathy initiated by exposed brain tissue factor. We examined an alternative hypothesis, namely, that marked release of tissue-type plasminogen activator (tPA) followed by delayed synthesis and release of urokinase plasminogen activator (uPA) from injured brain leads to posttraumatic bleeding by causing premature clot lysis. Using a murine model of severe TBI, we found that ICH is reduced in tPA(-/-) and uPA(-/-) mice but increased in PAI-1(-/-) mice compared with wild-type (WT) mice. tPA(-/-), but not uPA(-/-), mice developed a systemic coagulopathy post-TBI. Tranexamic acid inhibited ICH expansion in uPA(-/-)mice but not in tPA(-/-) mice. Catalytically inactive tPA-S(481)A inhibited plasminogen activation by tPA and uPA, attenuated ICH, lowered plasma d-dimers, lessened thrombocytopenia, and improved neurologic outcome in WT, tPA(-/-), and uPA(-/-) mice. ICH expansion was also inhibited by tPA-S(481)A in WT mice anticoagulated with warfarin. These data demonstrate that protracted endogenous fibrinolysis induced by TBI is primarily responsible for persistent ICH and post-TBI coagulopathy in this model and offer a novel approach to interrupt bleeding.

PAI-1-derived peptide EEIIMD prevents hypoxia/ischemia-induced aggravation of endothelin- and thromboxane-induced cerebrovasoconstriction.

BACKGROUND: Babies are frequently exposed to cerebral hypoxia and ischemia (H/I) during the perinatal period as a result of stroke, problems with delivery or post delivery respiratory management. The sole FDA approved treatment for acute stroke is tissue-type plasminogen activator (tPA). Endogenous tPA is upregulated and potentiates impairment of pial artery dilation in response to hypotension after H/I in pigs. Mitogen-activated protein kinase (MAPK), a family of at least 3 kinases, ERK, p38 and JNK, is also upregulated after H/I, with ERK contributing to impaired vasodilation. This study examined the hypothesis that H/I aggravates the vascular response to two important procontractile mediators released during CNS ischemia, endothelin-1 (ET-1) and thromboxane, which is further enhanced by tPA and ERK MAPK. METHODS: Cerebral hypoxia (pO(2) 35 mmHg for 10 min via inhalation of N(2)) followed immediately by ischemia (global intracranial pressure elevation for 20 min) was produced in chloralose anesthetized piglets equipped with a closed cranial window. RESULTS: H/I aggravated pial artery vasconstriction induced by ET-1 and the thromboxane mimetic U 46619. Potentiated vasoconstrictor responses were blocked by EEIIMD, an inhibitor of tPA's signaling and vascular activities, but unchanged by its inactive analogue EEIIMR. The cerebrospinal fluid concentration of ERK MAPK determined by ELISA was increased by H/I, potentiated by tPA, but blocked by EEIIMD. The ERK MAPK antagonist U 0126 blocked H/I augmented enhancement of ET-1 and U 46619 vasoconstriction. CONCLUSIONS: These data indicate that H/I aggravates ET-1 and thromboxane mediated cerebral vasoconstriction by upregulating endogenous tPA and ERK MAPK.

The plasminogen activator system: involvement in central nervous system inflammation and a potential site for therapeutic intervention.

BACKGROUND: Extracellular proteases such as plasminogen activators (PAs) and matrix metalloproteinases modulate cell-cell and cell-matrix interactions. Components of the PA/plasmin system have been shown to be increased in areas of inflammation, and have been suggested to play a role in inflammatory neurologic disorders such as epilepsy, stroke, brain trauma, Alzheimer's' disease and multiple sclerosis (MS). In the present study, we evaluated the involvement of the PA system in the animal model of MS, experimental autoimmune encephalomyelitis (EAE). METHODS: EAE was induced by myelin oligodendrocyte glycoprotein (MOG) in mice deficient for the urokinase PA (uPA-/-), or the urokinase PA receptor (uPAR-/-). Mice were evaluated for EAE clinical signs and histopathologic parameters, and compared with wild-type (WT) EAE mice. Lymphocytes from the knockout (KO) and WT mice were analyzed for ex vivo restimulation, cytokine secretion, and antigen presentation. Finally, WT EAE mice were treated with PAI-1dp, an 18 amino acid peptide derived from the PA inhibitor protein (PAI-1). RESULTS: EAE was aggravated in uPA-/- and uPAR-/- mice, and this was accompanied by more severe histopathologic features and microglial activation. By contrast, specific T- cell reactivity towards the encephalitogenic antigen MOG was markedly reduced in the KO animals, as shown by a marked reduction in proliferation and pro-inflammatory cytokine secretion in these mice. Antigen presentation was also reduced in all the KO animals, raising an immunologic paradox. When the mice were treated with PAI-1, a peptide derived from the PA system, a marked and significant improvement in EAE was seen. The clinical improvement was linked to reduced T-cell reactivity, further emphasizing the importance of the PA system in immunomodulation during neuroinflammation. CONCLUSIONS: Cumulatively, our results suggest a role for uPA and uPAR in EAE pathogenesis, as exacerbation of disease was seen in their absence. Furthermore, the successful amelioration of EAE by PAI-1 treatment suggests that the PA system can be considered a potential site for therapeutic intervention in the treatment of neuroimmune diseases.

tPA-S(481)A prevents impairment of cerebrovascular autoregulation by endogenous tPA after traumatic brain injury by upregulating p38 MAPK and inhibiting ET-1.

Traumatic brain injury (TBI) is associated with loss of cerebrovascular autoregulation, which leads to cerebral hypoperfusion. Mitogen activated protein kinase (MAPK) isoforms ERK, p38, and JNK and endothelin-1 (ET-1) are mediators of impaired cerebral hemodynamics after TBI. Excessive tissue plasminogen activator (tPA) released after TBI may cause loss of cerebrovascular autoregulation either by over-activating N-methyl-D-aspartate receptors (NMDA-Rs) or by predisposing to intracranial hemorrhage. Our recent work shows that a catalytically inactive tPA variant (tPA-S(481)A) that competes with endogenous wild type (wt) tPA for binding to NMDA-R through its receptor docking site but that cannot activate it, prevents activation of ERK by wt tPA and impairment of autoregulation when administered 30 min after fluid percussion injury (FPI). We investigated the ability of variants that lack proteolytic activity but bind/block activation of NMDA-Rs by wt tPA (tPA-S(481)A), do not bind/block activation of NMDA-Rs but are proteolytic (tPA-A(296-299)), or neither bind/block NMDA-Rs nor are proteolytic (tPA-A(296-299)S(481)A) to prevent impairment of autoregulation after TBI and the role of MAPK and ET-1 in such effects. Results show that tPA-S(481)A given 3 h post-TBI, but not tPA-A(296-299) or tPA-A(296-299)S(481)A prevents impaired autoregulation by upregulating p38 and inhibiting ET-1, suggesting that tPA-S(481)A has a realistic therapeutic window and focuses intervention on NMDA-Rs to improve outcome.

Combination therapy with glucagon and a novel plasminogen activator inhibitor-1-derived peptide enhances protection against impaired cerebrovasodilation during hypotension after traumatic brain injury through inhibition of ERK and JNK MAPK.

OBJECTIVE: Outcome of traumatic brain injury (TBI) is impaired by hypotension and glutamate, and TBI-associated release of endogenous tissue plasminogen activator (tPA) impairs cerebral autoregulation. Glucagon decreases central nervous system glutamate, lessens neuronal cell injury, and improves neurological score in mice after TBI. Glucagon partially protects against impaired cerebrovasodilation during hypotension after TBI in piglets by upregulating cAMP which decreases release of tPA. Pial artery dilation during hypotension is due to release of cAMP-dependent dilator prostaglandins (PG), such as PGE2 and PGI2. TBI impairs PGE2 and PGI2-mediated pial artery dilation, which contributes to disturbed cerebral autoregulation post-insult, by upregulating mitogen-activated protein kinase (MAPK). This study was designed to investigate relationships between tPA, prostaglandins, and MAPK as a mechanism to improve the efficacy of glucagon-mediated preservation of cerebrovasodilation during hypotension after TBI. METHODS: Lateral fluid percussion brain injury (FPI) was induced in piglets equipped with a closed cranial window. ERK and JNK MAPK concentrations in cerebrospinal fluid were quantified by enzyme-linked immunosorbent assay. RESULTS: Cerebrospinal fluid JNK MAPK was increased by FPI, but blunted by glucagon and the novel plasminogen activator inhibitor-1-derived peptide (PAI-1DP), Ac-RMAPEEIIMDRPFLYVVR-amide. FPI modestly increased, while glucagon and PAI-1DP decreased ERK MAPK. PGE2, PGI2, N-methyl-D-aspartate, and hypotension-induced pial artery dilation was blunted after FPI, partially protected by glucagon, and fully protected by glucagon+PAI-1DP, glucagon+JNK antagonist SP600125 or glucagon+ERK inhibitor U 0126. DISCUSSION: Glucagon+PAI-1DP act in concert to protect against impairment of cerebrovasodilation during hypotension after TBI via inhibition of ERK and JNK MAPK.

tPA-S481A prevents neurotoxicity of endogenous tPA in traumatic brain injury.

Traumatic brain injury (TBI) is associated with loss of autoregulation due to impaired responsiveness to cerebrovascular dilator stimuli, which leads to cerebral hypoperfusion and neuronal impairment or death. Upregulation of tissue plasminogen activator (tPA) post-TBI exacerbates loss of cerebral autoregulation and NMDA-receptor-mediated impairment of cerebral hemodynamics, and enhances excitotoxic neuronal death. However, the relationship between NMDA-receptor activation, loss of autoregulation, and neurological dysfunction is unclear. Here, we evaluated the potential therapeutic efficacy of a catalytically inactive tPA variant, tPA S481A, that acts by competing with wild-type tPA for binding, cleavage, and activation of NMDA receptors. Lateral fluid percussion brain injury was produced in anesthetized piglets. Pial artery reactivity was measured via a closed cranial window, and cerebrospinal fluid (CSF) extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) was quantified by enzyme-linked immunosorbent assay (ELISA). tPA-S481A prevented impairment of cerebral autoregulation and reduced histopathologic changes after TBI by inhibiting upregulation of the ERK isoform of MAPK. Treatment with this tPA variant provides a novel approach for limiting neuronal toxicity caused by untoward NMDA-receptor activation mediated by increased tPA and glutamate following TBI.

RBC-coupled tPA Prevents Whereas tPA Aggravates JNK MAPK-Mediated Impairment of ATP- and Ca-Sensitive K Channel-Mediated Cerebrovasodilation After Cerebral Photothrombosis.

The sole Food and Drug Administration-approved treatment for acute stroke is tissue-type plasminogen activator (tPA), but tPA aggravates impairment of cerebrovasodilation during hypotension in a newborn pig photothrombotic model of stroke. Coupling to carrier red blood cells (RBC) enhances thrombolytic effects of tPA, while reducing its side effects. ATP- and Ca-sensitive K channels (Katp and Kca) are important regulators of cerebrovascular tone and mediate cerebrovasodilation during hypotension. Mitogen-activated protein kinase, a family of at least three kinases, ERK, p38, and c-Jun-N-terminal kinase (JNK), is upregulated after photothrombosis. This study examined the effect of photothrombosis on Katp- and Kca-induced cerebrovasodilation and the roles of tPA and JNK during/after injury. Photothrombosis blunted vasodilation induced by the Katp agonists cromakalim, calcitonin gene-related peptide, and the Kca agonist NS 1619, which was aggravated by injection of tPA. In contrast, both pre- or post-injury thrombosis injection of RBC-tPA and JNK antagonist SP 600125 prevented impairment of Katp- and Kca-induced vasodilation. Therefore, JNK activation in thrombosis impairs K channel-mediated cerebrovasodilation. Standard thrombolytic therapy of central nervous system ischemic disorders using free tPA poses the danger of further dysregulation of cerebrohemodynamics by impairing cation-mediated control of cerebrovascular tone, whereas RBC-coupled tPA both restores reperfusion and normalizes cerebral hemodynamics.