Ya Ching Hsieh

Tissue-specific expression of estrogen receptors and their role in the regulation of neutrophil infiltration in various organs following trauma-hemorrhage

Although 17β‐estradiol (E2) administration after trauma‐hemorrhage (T‐H) reduces tissue neutrophil sequestration in male rodents, it remains unknown which of the estrogen receptor (ER) subtypes mediates this effect and whether the same ER subtype is involved in all the tissues. We hypothesized that the salutary effects of E2 on attenuation of neutrophil accumulation following T‐H are tissue and receptor subtype‐specific. Male Sprague‐Dawley rats underwent sham operation or T‐H (mean blood pressure, 40 mmHg for 90 min and then resuscitation). E2 (50 μg/kg), ER‐α agonist propyl pyrazole triol (PPT; 5 μg/kg), ER‐β agonist diarylpropiolnitrile (DPN; 5 μg/kg), or vehicle (10% dimethyl sulfoxide) was administered subcutaneously during resuscitation. Twenty‐four hours thereafter, tissue myeloperoxidase (MPO) activity (a marker of neutrophil sequestration), cytokine‐induced neutrophil chemoattractant (CINC)‐1, CINC‐3, and intercellular adhesion molecule (ICAM)‐1 levels in the liver, intestine, and lung were measured (n=6 rats/group). ER‐α and ER‐β mRNA levels in sham‐operated rats were also determined. T‐H increased MPO activity, CINC‐1, CINC‐3, and ICAM‐1 levels in the liver, intestine, and lung. These parameters were improved significantly in rats receiving E2 after T‐H. Administration of the ER‐α agonist PPT but not the ER‐β agonist DPN improved the measured parameters in the liver. In contrast, DPN but not PPT significantly improved these parameters in the lung. In the intestine, ER subtype specificity was not observed. ER‐α mRNA expression was highest in the liver, whereas ER‐β mRNA expression was greatest in the lung. Thus, the salutary effects of E2 administration on tissue neutrophil sequestration following T‐H are receptor subtype and tissue‐specific.

The PI3K/Akt Pathway Mediates the Nongenomic Cardioprotective Effects of Estrogen Following Trauma-hemorrhage

Objective:To determine whether the nongenomic actions of E2 have any beneficial effect on cardiac function following trauma-hemorrhage and whether those effects are mediated via the PI3K/Akt pathway. Summary Background Data:Since studies suggest that both genomic and nongenomic pathways are involved in mediating the salutary effects of 17β-estradiol (estradiol) following trauma-hemorrhage, we examined if the nongenomic effects of estradiol on cardiac function after trauma-hemorrhage involve the PI3K/Akt pathway. Methods:Male Sprague-Dawley rats (∼300 g) underwent trauma-hemorrhage (mean blood pressure, 40 mm Hg for 90 min, then resuscitation). Estradiol conjugated to bovine serum albumin (BSA) (estradiol-BSA; 1 mg/kg estradiol) with or without estrogen receptor antagonist (ICI 182,780), PI3K inhibitor (Wortmannin), or vehicle was injected intravenously during resuscitation. At 2 hours after trauma-hemorrhage or sham operation, cardiac output, stroke volume, heart rate, mean arterial pressure, and ±dP/dt were measured. Cardiomyocyte PI3K, p-Akt, Akt protein expressions and apoptosis were also determined. One-way ANOVA and Tukeys test were used for statistical analysis. Results:Cardiac output, stroke volume, and ±dP/dt decreased significantly after trauma-hemorrhage. Administration of estradiol or estradiol-BSA significantly improved these parameters of cardiac function. Although trauma-hemorrhage decreased cardiomyocyte PI3K protein expression and Akt phosphorylation (p-Akt), estradiol or estradiol-BSA treatment following trauma-hemorrhage prevented such decreases in cardiomyocyte PI3K protein expressions and p-Akt. The increase in cardiomyocyte apoptosis was also prevented in rats receiving estradiol-BSA. Co-administration of ICI 182,780 or Wortmannin abolished beneficial effects of estradiol-BSA on cardiac functions following trauma-hemorrhage. Conclusion:The PI3K/Akt pathway plays a critical role in mediating the nongenomic salutary effects of estradiol on cardiac function following trauma-hemorrhage.

The Role of MIP-1α in the Development of Systemic Inflammatory Response and Organ Injury following Trauma Hemorrhage

Although MIP-1α is an important chemokine in the recruitment of inflammatory cells, it remains unknown whether MIP-1α plays any role in the development of systemic inflammatory response following trauma-hemorrhage (T-H). C57BL/6J wild type (WT) and MIP-1α-deficient (KO) mice were used either as control, subjected to sham operation (cannulation or laparotomy only or cannulation plus laparotomy) or T-H (midline laparotomy, mean blood pressure 35 ± 5 mmHg for 90 min, followed by resuscitation) and sacrificed 2 h thereafter. A marked increase in serum α-glutathione transferase, TNF-α, IL-6, IL-10, MCP-1, and MIP-1α and Kupffer cell cytokine production was observed in WT T-H mice compared with shams or control. In addition lung and liver tissue edema and neutrophil infiltration (myeloperoxidase (MPO) content) was also increased following T-H in WT animals. These inflammatory markers were markedly attenuated in the MIP-1α KO mice following T-H. Furthermore, compared with 2 h, MPO activities at 24 and 48 h after T-H declined steadily in both WT and KO mice. However, normalization of MPO activities to sham levels within 24 h was seen in KO mice but not in WT mice. Thus, MIP-1α plays an important role in mediating the acute inflammatory response following T-H. In the absence of MIP-1α, acute inflammatory responses were attenuated; rapidly recovered and less remote organ injury was noted following T-H. Thus, interventions that reduce MIP-1α levels following T-H should be useful in decreasing the deleterious inflammatory consequence of trauma.

Keratinocyte-derived chemokine plays a critical role in the induction of systemic inflammation and tissue damage after trauma-hemorrhage

Neutrophil infiltration is a crucial step in the development of organ dysfunction after trauma. We have previously shown that keratinocyte-derived chemokine (KC), a chemoattractant for neutrophils, is up-regulated after trauma-hemorrhage. To determine the role of KC after trauma-hemorrhage, the effect of a KC-neutralizing antibody on the posttraumatic inflammatory response was examined. One hour before surgery, male C3H/HeN mice were treated with an anti-KC antibody or isotype control. Animals were subjected to sham operation or trauma-hemorrhage and resuscitated with Ringer lactate thereafter. They were killed 2 h later, and Kupffer cells were isolated. Plasma levels, Kupffer cell production, and lung and liver content of TNF-α, IL-6, IL-10, monocyte chemoattractant protein 1, macrophage inflammatory protein 1α, and KC were determined by BD cytometric bead arrays. Myeloperoxidase content in lung and liver were measured as a parameter for neutrophil infiltration, and wet-to-dry weight ratios of these organs were also determined. Hepatocyte damage was assessed by measuring α-gluthathione S-transferase concentration. Administration of the anti-KC antibody before trauma-hemorrhage prevented increases in KC plasma levels, which was accompanied by amelioration of neutrophil infiltration and edema formation in lung and liver after trauma-hemorrhage. No effect on other cytokines in plasma or Kupffer cell release was observed. These results suggest that KC plays a pivotal role in neutrophil infiltration and organ damage after trauma-hemorrhage and resuscitation.

Mechanism of the salutary effects of flutamide on intestinal myeloperoxidase activity following trauma-hemorrhage: up-regulation of estrogen receptor-β-dependent HO-1

Hemeoxygenase (HO)‐1 induction following adverse circulatory conditions is known to be protective, and precastrated males have less intestinal damage than sham‐operated males following trauma‐hemorrhage (T‐H). Previous studies have also shown that administration of flutamide up‐regulated estrogen receptor (ER) expression in males following T‐H. We hypothesized that flutamide administration in males following T‐H up‐regulates HO‐1 via an ER‐dependent pathway and protects against intestinal injury. Male Sprague‐Dawley rats underwent T‐H [mean blood pressure (MBP) 40 mmHg for 90 min and then resuscitation]. A single dose of flutamide (25 mg/kg body weight), with or without an ER antagonist (ICI 182,780), a HO enzyme inhibitor [chromium‐mesoporphyrin (CrMP)], or vehicle, was administered subcutaneously during resuscitation. At 2 h after T‐H or sham operation, intestinal myeloperoxidase (MPO) activity, intercellular adhesion molecule (ICAM)‐1, cytokine‐induced neutrophil chemoattractant (CINC)‐1, and CINC‐3 levels were measured. Intestinal ER‐α, ER‐β, androgen receptor, and HO‐1 mRNA/protein levels were also determined. Results showed that T‐H increased intestinal MPO activity, ICAM‐1, CINC‐1, and CINC‐3 levels. These parameters were improved significantly in the flutamide‐treated rats subjected to T‐H. Flutamide treatment increased intestinal HO‐1 and ER‐β mRNA/protein levels as compared with vehicle‐treated T‐H rats. Administration of the ER antagonist ICI 182,780 or the HO inhibitor CrMP prevented the flutamide‐induced attenuation of shock‐induced intestinal damage. Thus, the salutary effects of flutamide administration on attenuation of intestinal injury following T‐H are mediated via up‐regulation of ER‐β‐dependent HO‐1 expression.

Mechanism of the nongenomic effects of estrogen on intestinal myeloperoxidase activity following trauma-hemorrhage: up-regulation of the PI-3K/Akt pathway

As studies indicate that genomic and nongenomic pathways are involved in mediating the salutary effects of 17β‐estradiol (E2) following trauma‐hemorrhage, we examined if the nongenomic effects of E2 on attenuation of intestinal injury after trauma‐hemorrhage involve the PI‐3K/Akt pathway. Male Sprague‐Dawley rats (∼300 g body weight) underwent trauma‐hemorrhage (mean blood pressure 40 mmHg for 90 min), followed by resuscitation. E2 conjugated to BSA (E2‐BSA; 1 mg/Kg E2), with or without an estrogen receptor antagonist (ICI 182,780), a PI‐3K inhibitor (Wortmannin), or vehicle, was injected i.v. during resuscitation. At 2 h after trauma‐hemorrhage or sham operation, intestinal myeloperoxidase (MPO) activity, ICAM‐1, cytokine‐induced neutrophil chemoattractant (CINC)‐1, CINC‐3, and IL‐6 levels were measured (n=6 rats/group). Intestinal PI‐3K, phosphorylation of Akt (p‐Akt), and Akt protein expressions were also determined. One‐way ANOVA and Tukeys test were used for statistical analysis. The results indicated that trauma‐hemorrhage increased intestinal MPO activity and ICAM‐1, CINC‐1, CINC‐3, and IL‐6 levels. These parameters were improved significantly in the E2‐ or E2‐BSA‐treated rats subjected to trauma‐hemorrhage. Although trauma‐hemorrhage decreased intestinal PI‐3K and p‐Akt protein expressions, E2 or E2‐BSA treatment following trauma‐hemorrhage prevented such decreases in intestinal PI‐3K and p‐Akt protein expressions. Coadministration of ICI 182,780 or Wortmannin abolished the beneficial effects of E2‐BSA on attenuation of intestinal injury following trauma‐hemorrhage. Thus, the PI‐3K/Akt pathway plays a critical role in mediating the nongenomic, salutary effects of E2 on attenuation of shock‐induced intestinal tissue damage.

Downregulation of TLR4-dependent ATP production is critical for estrogen-mediated immunoprotection in Kupffer cells following trauma-hemorrhage.

Toll‐like receptor 4 (TLR4) mediates mitochondrial DNA (mtDNA) damage and biogenic responses. Mitochondrial transcription factor A (Tfam) is an essential regulator for mtDNA transcription and ATP production. Increased ATP levels were associated with normalization of immune function following trauma‐hemorrhage. Moreover, administration of 17β‐estradiol following trauma‐hemorrhage upregulates cardiac Tfam and ATP levels. We therefore hypothesized that the salutary effect of 17β‐estradiol on Kupffer cell function following trauma‐hemorrhage is mediated via negative regulation of TLR4, which downregulates iNOS, upregulates Tfam and mtDNA‐encoded gene cytochrome c oxidase I (mtCOI), and consequently increases cellular ATP levels. Male C3H/HeN, C3H/HeOuJ (intact TLR4), and C3H/HeJ (TLR4 mutant) mice were subjected to trauma‐hemorrhage (mean BP 35u2009±u20095 mmHg ∼90 min, then resuscitation) or sham operation. At the beginning of resuscitation, mice received 17β‐estradiol (25 µg/25 g) or vehicle intravenously and were sacrificed 2 h thereafter. Kupffer cell TLR4, iNOS, IL‐6 and TNF‐α production capacities were increased, and ATP, Tfam, and mtCOI levels were decreased following trauma‐hemorrhage. Administration of 17β‐estradiol following trauma‐hemorrhage prevented the increase in Kupffer cell TLR4, iNOS, and cytokine production. This was accompanied by normalized ATP, Tfam, and mtCOI levels. Furthermore, the decreased Kupffer cell ATP and mtCOI levels were not observed in TLR4 mutant mice following trauma‐hemorrhage. Taken together, these findings suggest that downregulation of TLR4‐dependent ATP production is critical to 17β‐estradiol‐mediated immunoprotection in Kupffer cells following trauma‐hemorrhage. J. Cell. Physiol. 211: 364–370, 2007.

MECHANISM OF ESTROGEN-MEDIATED IMPROVEMENT IN CARDIAC FUNCTION AFTER TRAUMA-HEMORRHAGE: p38-DEPENDENT NORMALIZATION OF CARDIAC Akt PHOSPHORYLATION AND GLYCOGEN LEVELS

Both p38 mitogen-activated protein kinase (p38) activation and protein kinase B (Akt) activation have been reported to regulate glucose transport during myocardial I/R. An increase in cardiac glycogen levels prevents myocardial injury in the ischemic or stressed heart. Although studies have shown that 17&bgr;-estradiol (E2)-mediated improvement in cardiac function after trauma-hemorrhage is via p38 activation, it remains unknown whether p38/Akt plays any role in regulation of cardiac glycogen levels under these conditions. To study this, male rats underwent trauma-hemorrhage (mean blood pressure, ×40 mmHg for 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats (n = 6 per group) were treated with vehicle, E2 (1 mg/kg body weight), the p38 inhibitor SB203580 (2 mg/kg body weight), or E2 and SB203580. Various parameters were measured at 2 h after resuscitation. One-way ANOVA and Tukey test were used for statistical analysis, and differences were considered significant at P < 0.05. The depressed cardiac function after trauma-hemorrhage was restored by E2 treatment (P < 0.05). Administration of E2 after trauma-hemorrhage also normalized the p38/Akt phosphorylation, which was associated with restoration of cardiac glycogen, glycogen synthase kinase 3&bgr; activation, glucose transporter 4 translocation, and increased hexokinase II levels (all parameters, P < 0.05). Inhibition of the p38 pathway abolished the E2-induced restoration in above parameters after trauma-hemorrhage. These results suggest that p38-dependent normalization of cardiac Akt phosphorylation and glycogen levels plays an important role in E2-mediated restoration of cardiac function after trauma-hemorrhage.

Mechanism of salutary effects of finasteride on post-traumatic immune/inflammatory response: upregulation of estradiol synthesis.

Objective:The aim of this study was to evaluate whether pretreatment with finasteride, a 5α-reductase inhibitor, improves immune functions after trauma-hemorrhage. Summary Background Data:A number of studies have provided evidence for a gender dimorphism in host defense after trauma. Under stress conditions, such as trauma-hemorrhage, androgenic hormones have immunosuppressive effects, leading to increased susceptibility to sepsis, morbidity, and mortality. Testosterone is converted by 5α-reductase to 5α-dihydrotestosterone (DHT), a more potent androgen. Methods:Male C3H/HeN mice (8–10 weeks) were randomly assigned to receive finasteride or vehicle for 2 days and were then subjected to trauma-hemorrhage or sham operation. Trauma-hemorrhage was induced by a midline laparotomy and approximately 90 minutes of hemorrhagic shock (blood pressure, 35 mm Hg), followed by fluid resuscitation. Animals were killed 2 hours after resuscitation or sham procedure. Plasma levels and Kupffer cell production of cytokines (TNF-α, IL-6, IL-10, MCP-1, KC, and MIP-1α), lung neutrophil infiltration, and edema were evaluated. Results:Finasteride administration prevented the increase in cytokine plasma levels, decreased DHT, and increased 17β-estradiol plasma concentrations. In addition, neutrophil infiltration and edema formation in the lung were reduced by finasteride. The salutary effects of finasteride were abrogated after coadministration with an estrogen receptor inhibitor (ICI 182,780). Increased Kupffer cell cytokine production normally observed after trauma-hemorrhage was prevented by treatment with finasteride. Conclusion:These results suggest that inhibition of 5α-reductase leads to the conversion of testosterone to 17β-estradiol, which produces salutary effects on the post-traumatic immune response.

Suppression of Activation and Costimulatory Signaling in Splenic CD4 T Cells after Trauma-Hemorrhage Reduces T-Cell Function A Mechanism of Post-Traumatic Immune Suppression

Reduced immune function is frequently a consequence of serious injury such as trauma-hemorrhage (T-H). Injury may lead to reduced T-cell activation, resulting in decreased engagement of costimulatory molecules after antigen recognition and in subsequent immunological compromise and anergy. We hypothesized that inhibition of CD28 expression is one possible mechanism by which immune functions are suppressed after T-H. Male C3H/HeN mice (with or without ovalbumin immunization) were subjected to sham operation or T-H and sacrificed after 24 hours. Splenic T cells were then stimulated with concanavalin A or ovalbumin in vivo or in vitro, and CD28, cytotoxic T-lymphocyte antigen 4 (CTLA-4), CD69, and phospho-Akt expression was determined. T-cell proliferation/cytokine production was measured in vitro. Stimulation-induced CD69, CD28, and phospho-Akt up-regulation were significantly impaired after T-H compared with sham-operated animals; however, CTLA-4 expression was significantly higher in the T-H group. Over a 3-day span, stimulated T cells from sham-operated animals showed significantly higher proliferation compared with the T-H group. IL-2 and IFN-gamma were elevated in sham-operated animals, whereas IL-4 and IL-5 rose in the T-H group, revealing a shift from T(H)1 to T(H)2 type cytokine production after T-H. Dysregulation of the T-cell costimulatory pathway is therefore likely to be a significant contributor to post-traumatic immune suppression.