Time-Restricted Salutary Effects of Blood Flow Restoration on Venous Thrombosis and Vein Wall Injury in Mouse and Human Subjects.

BACKGROUND: Up to 50% of patients with proximal deep vein thrombosis (DVT) will develop the postthrombotic syndrome characterized by limb swelling and discomfort, hyperpigmentation, skin ulcers, and impaired quality of life. Although catheter-based interventions enabling the restoration of blood flow (RBF) have demonstrated little benefit on postthrombotic syndrome, the impact on the acuity of the thrombus and mechanisms underlying this finding remain obscure. In experimental and clinical studies, we examined whether RBF has a restricted time window for improving DVT resolution. METHODS: First, experimental stasis DVT was generated in C57/BL6 mice (n=291) by inferior vena cava ligation. To promote RBF, mice underwent mechanical deligation with or without intravenous recombinant tissue plasminogen activator administered 2 days after deligation. RBF was assessed over time by ultrasonography and intravital microscopy. Resected thrombosed inferior vena cava specimens underwent thrombus and vein wall histological and gene expression assays. Next, in a clinical study, we conducted a post hoc analysis of the ATTRACT (Acute Venous Thrombosis: Thrombus Removal with Adjunctive Catheter-Directed Thrombolysis) pharmacomechanical catheter-directed thrombolysis (PCDT) trial (NCT00790335) to assess the effects of PCDT on Venous Insufficiency Epidemiological and Economic Study quality-of-life and Villalta scores for specific symptom-onset-to-randomization timeframes. RESULTS: Mice that developed RBF by day 4, but not later, exhibited reduced day 8 thrombus burden parameters and reduced day 8 vein wall fibrosis and inflammation, compared with controls. In mice without RBF, recombinant tissue plasminogen activator administered at day 4, but not later, reduced day 8 thrombus burden and vein wall fibrosis. It is notable that, in mice already exhibiting RBF by day 4, recombinant tissue plasminogen activator administration did not further reduce thrombus burden or vein wall fibrosis. In the ATTRACT trial, patients receiving PCDT in an intermediate symptom-onset-to-randomization timeframe of 4 to 8 days demonstrated maximal benefits in Venous Insufficiency Epidemiological and Economic Study quality-of-life and Villalta scores (between-group difference=8.41 and 1.68, respectively, P<0.001 versus patients not receiving PCDT). PCDT did not improve postthrombotic syndrome scores for patients having a symptom-onset-to-randomization time of <4 days or >8 days. CONCLUSIONS: Taken together, these data illustrate that, within a restricted therapeutic window, RBF improves DVT resolution, and PCDT may improve clinical outcomes. Further studies are warranted to examine the value of time-restricted RBF strategies to reduce postthrombotic syndrome in patients with DVT.

Venous stasis-induced fibrinolysis prevents thrombosis in mice: role of α2-antiplasmin.

Stasis of venous blood triggers deep vein thrombosis by activating coagulation, yet its effects on the fibrinolytic system are not fully understood. We examined the relationship between stasis, fibrinolysis, and the development of experimental venous thrombosis. Effects of stasis-induced deep vein thrombosis and fibrinolysis on thrombosis were examined by inferior vena cava ligation in congenic mice with and without α2-antiplasmin (α2AP), the primary inhibitor of plasmin. Venous thrombus weights were measured and thrombus composition was determined by Martius scarlet blue and immunofluorescence staining. Venous thrombi from α2AP+/+ mice contained plasminogen activators, plasminogen activator inhibitor-1, plasminogen, and α2AP, which changed with thrombus age. Normal, α2AP+/+ mice developed large, occlusive thrombi within 5 hours after ligation; thrombi were even larger in plasminogen-deficient mice (P < .001). No significant thrombus formation was seen in α2AP-/- mice (P < .0001) or in α2AP+/+ mice treated with an α2AP-inactivating antibody (P < .001). Venous stasis activated fibrinolysis, measured by D-dimer levels, in α2AP-/- mice vs α2AP+/+ mice (P < .05). Inhibition of fibrinolysis by the indirect plasmin inhibitor ε-aminocaproic acid or by α2AP restored thrombosis in α2AP-/- mice. In addition to its effects on acute thrombosis, thrombus formation was also markedly suppressed in α2AP-/- mice vs α2AP+/+ mice (P < .0001) 1, 7, and 14 days after ligation. We conclude that experimental venous stasis activates the fibrinolytic system to block the development of venous thrombosis. Suppression of fibrinolysis by α2AP appears essential for stasis-induced thrombus development, which suggests that targeting α2AP may prove useful for preventing venous thrombosis.

A Low-Sodium Diet Boosts Ang (1-7) Production and NO-cGMP Bioavailability to Reduce Edema and Enhance Survival in Experimental Heart Failure.

Sodium restriction is often recommended in heart failure (HF) to block symptomatic edema, despite limited evidence for benefit. However, a low-sodium diet (LSD) activates the classical renin-angiotensin-aldosterone system (RAAS), which may adversely affect HF progression and mortality in patients with dilated cardiomyopathy (DCM). We performed a randomized, blinded pre-clinical trial to compare the effects of a normal (human-equivalent) sodium diet and a LSD on HF progression in a normotensive model of DCM in mice that has translational relevance to human HF. The LSD reduced HF progression by suppressing the development of pleural effusions (p < 0.01), blocking pathological increases in systemic extracellular water (p < 0.001) and prolonging median survival (15%, p < 0.01). The LSD activated the classical RAAS by increasing plasma renin activity, angiotensin II and aldosterone levels. However, the LSD also significantly up-elevated the counter-regulatory RAAS by boosting plasma angiotensin converting enzyme 2 (ACE2) and angiotensin (1-7) levels, promoting nitric oxide bioavailability and stimulating 3'-5'-cyclic guanosine monophosphate (cGMP) production. Plasma HF biomarkers associated with poor outcomes, such as B-type natriuretic peptide and neprilysin were decreased by a LSD. Cardiac systolic function, blood pressure and renal function were not affected. Although a LSD activates the classical RAAS system, we conclude that the LSD delayed HF progression and mortality in experimental DCM, in part through protective stimulation of the counter-regulatory RAAS to increase plasma ACE2 and angiotensin (1-7) levels, nitric oxide bioavailability and cGMP production.

In Experimental Dilated Cardiomyopathy Heart Failure and Survival Are Adversely Affected by a Lack of Sexual Interactions.

Nearly one in three people in the U.S. will develop heart failure (HF), characterized by fluid retention (edema) in the lungs and elsewhere. This leads to difficult breathing, deterioration of physical capacity, restriction of normal activities and death. There is little data about the safety and effects of sexual interactions in patients with HF. We tested whether a lack of sexual interactions affected pathophysiological outcomes in a pre-clinical mouse model of dilated cardiomyopathy that recapitulates the progressive stages of human HF. Male mice were randomly given access to, or deprived from, sexual interactions with female mice, which were confirmed by videography and generation of offspring. Cohousing with access to sexual interactions markedly prolonged survival, while cohousing without access to sexual activity did not. Sexual interactions improved systolic function, reduced HF-associated edema, altered transcription of heart contractile protein genes and decreased plasma testosterone levels. To determine whether testosterone levels contributed to survival, testosterone levels were experimentally reduced. Reduction of testosterone levels significantly prolonged survival. Taken together, in mice with dilated cardiomyopathy, sexual activity altered cardiac contractile gene transcription, improved systolic function, reduced edema and prolonged survival which may be in part due to lower testosterone levels.

Corin Overexpression Reduces Myocardial Infarct Size and Modulates Cardiomyocyte Apoptotic Cell Death.

Altered expression of corin, a cardiac transmembrane serine protease, has been linked to dilated and ischemic cardiomyopathy. However, the potential role of corin in myocardial infarction (MI) is lacking. This study examined the outcomes of MI in wild-type vs. cardiac-specific overexpressed corin transgenic (Corin-Tg) mice during pre-MI, early phase (3, 24, 72 h), and late phase (1, 4 weeks) post-MI. Corin overexpression significantly reduced cardiac cell apoptosis (p < 0.001), infarct size (p < 0.001), and inhibited cleavage of procaspases 3, 9, and 8 (p < 0.05 to p < 0.01), as well as altered the expression of Bcl2 family proteins, Bcl-xl, Bcl2 and Bak (p < 0.05 to p < 0.001) at 24 h post-MI. Overexpressed cardiac corin also significantly modulated heart function (ejection fraction, p < 0.0001), lung congestion (lung weight to body weight ratio, p < 0.0001), and systemic extracellular water (edema, p < 0.05) during late phase post-MI. Overall, cardiac corin overexpression significantly reduced apoptosis, infarct size, and modulated cardiac expression of key members of the apoptotic pathway in early phase post-MI; and led to significant improvement in heart function and reduced congestion in late phase post-MI. These findings suggest that corin may be a useful target to protect the heart from ischemic injury and subsequent post-infarction remodeling.

Normalizing Plasma Renin Activity in Experimental Dilated Cardiomyopathy: Effects on Edema, Cachexia, and Survival.

Heart failure (HF) patients frequently have elevated plasma renin activity. We examined the significance of elevated plasma renin activity in a translationally-relevant model of dilated cardiomyopathy (DCM), which replicates the progressive stages (A-D) of human HF. Female mice with DCM and elevated plasma renin activity concentrations were treated with a direct renin inhibitor (aliskiren) in a randomized, blinded fashion beginning at Stage B HF. By comparison to controls, aliskiren treatment normalized pathologically elevated plasma renin activity (p < 0.001) and neprilysin levels (p < 0.001), but did not significantly alter pathological changes in plasma aldosterone, angiotensin II, atrial natriuretic peptide, or corin levels. Aliskiren improved cardiac systolic function (ejection fraction, p < 0.05; cardiac output, p < 0.01) and significantly reduced the longitudinal development of edema (extracellular water, p < 0.0001), retarding the transition from Stage B to Stage C HF. The normalization of elevated plasma renin activity reduced the loss of body fat and lean mass (cachexia/sarcopenia), p < 0.001) and prolonged survival (p < 0.05). In summary, the normalization of plasma renin activity retards the progression of experimental HF by improving cardiac systolic function, reducing the development of systemic edema, cachexia/sarcopenia, and mortality. These data suggest that targeting pathologically elevated plasma renin activity may be beneficial in appropriately selected HF patients.

Renin Activity in Heart Failure with Reduced Systolic Function-New Insights.

Regardless of the cause, symptomatic heart failure (HF) with reduced ejection fraction (rEF) is characterized by pathological activation of the renin-angiotensin-aldosterone system (RAAS) with sodium retention and extracellular fluid expansion (edema). Here, we review the role of active renin, a crucial, upstream enzymatic regulator of the RAAS, as a prognostic and diagnostic plasma biomarker of heart failure with reduced ejection fraction (HFrEF) progression; we also discuss its potential as a pharmacological bio-target in HF therapy. Clinical and experimental studies indicate that plasma renin activity is elevated with symptomatic HFrEF with edema in patients, as well as in companion animals and experimental models of HF. Plasma renin activity levels are also reported to be elevated in patients and animals with rEF before the development of symptomatic HF. Modulation of renin activity in experimental HF significantly reduces edema formation and the progression of systolic dysfunction and improves survival. Thus, specific assessment and targeting of elevated renin activity may enhance diagnostic and therapeutic precision to improve outcomes in appropriate patients with HFrEF.

Cardiac-Specific Overexpression of Catalytically Inactive Corin Reduces Edema, Contractile Dysfunction, and Death in Mice with Dilated Cardiomyopathy.

Humans with dilated cardiomyopathy (DCM) and heart failure (HF) develop low levels of corin, a multi-domain, cardiac-selective serine protease involved in natriuretic peptide cleavage and sodium and water regulation. However, experimental restoration of corin levels markedly attenuates HF progression. To determine whether the beneficial effects of corin in HF require catalytic activity, we engineered cardiac overexpression of an enzymatically inactive corin transgene (corin-Tg(i)). On a wild-type (WT) background, corin-Tg(i) had no evident phenotypic effects. However, in a well-established genetic model of DCM, corin-Tg(i)/DCM mice had increased survival (p < 0.01 to 0.001) vs. littermate corin-WT/DCM controls. Pleural effusion (p < 0.01), lung edema (p < 0.05), systemic extracellular free water (p < 0.01), and heart weight were decreased (p < 0.01) in corin-Tg(i)/DCM vs. corin-WT/DCM mice. Cardiac ejection fraction and fractional shortening improved (p < 0.01), while ventricular dilation decreased (p < 0.0001) in corin-Tg(i)/DCM mice. Plasma atrial natriuretic peptide, cyclic guanosine monophosphate, and neprilysin were significantly decreased. Cardiac phosphorylated glycogen synthase kinase-3ß (pSer9-GSK3ß) levels were increased in corin(i)-Tg/DCM mice (p < 0.01). In summary, catalytically inactive corin-Tg(i) decreased fluid retention, improved contractile function, decreased HF biomarkers, and diminished cardiac GSK3ß activity. Thus, the protective effects of cardiac corin on HF progression and survival in experimental DCM do not require the serine protease activity of the molecule.

Releasing the Brakes on the Fibrinolytic System in Pulmonary Emboli: Unique Effects of Plasminogen Activation and α2-Antiplasmin Inactivation.

BACKGROUND: In patients with hemodynamically significant pulmonary embolism, physiological fibrinolysis fails to dissolve thrombi acutely and r-tPA (recombinant tissue-type plasminogen activator) therapy may be required, despite its bleeding risk. To examine potential mechanisms, we analyzed the expression of key fibrinolytic molecules in experimental pulmonary emboli, assessed the contribution of α2-antiplasmin to fibrinolytic failure, and compared the effects of plasminogen activation and α2-antiplasmin inactivation on experimental thrombus dissolution and bleeding. METHODS: Pulmonary embolism was induced by jugular vein infusion of 125I-fibrin or fluorescein isothiocyanate-fibrin labeled emboli in anesthetized mice. Thrombus site expression of key fibrinolytic molecules was determined by immunofluorescence staining. The effects of r-tPA and α2-antiplasmin inactivation on fibrinolysis and bleeding were examined in a humanized model of pulmonary embolism. RESULTS: The plasminogen activation and plasmin inhibition system assembled at the site of acute pulmonary emboli in vivo. Thrombus dissolution was markedly accelerated in mice with normal α2-antiplasmin levels treated with an α2-antiplasmin-inactivating antibody (P<0.0001). Dissolution of pulmonary emboli by α2-antiplasmin inactivation alone was comparable to 3 mg/kg r-tPA. Low-dose r-tPA alone did not dissolve emboli, but was synergistic with α2-antiplasmin inactivation, causing more embolus dissolution than clinical-dose r-tPA alone (P<0.001) or α2-antiplasmin inactivation alone (P<0.001). Despite greater thrombus dissolution, α2-antiplasmin inactivation alone, or in combination with low-dose r-tPA, did not lead to fibrinogen degradation, did not cause bleeding (versus controls), and caused less bleeding than clinical-dose r-tPA (P<0.001). CONCLUSIONS: Although the fibrinolytic system assembles at the site of pulmonary emboli, thrombus dissolution is halted by α2-antiplasmin. Inactivation of α2-antiplasmin was comparable to pharmacological r-tPA for dissolving thrombi. However, α2-antiplasmin inactivation showed a unique pattern of thrombus specificity, because unlike r-tPA, it did not degrade fibrinogen or enhance experimental bleeding. This suggests that modifying the activity of a key regulator of the fibrinolytic system, like α2-antiplasmin, may have unique therapeutic value in pulmonary embolism.

α2-Antiplasmin: New Insights and Opportunities for Ischemic Stroke.

Thrombotic vascular occlusion is the leading cause of ischemic stroke. High blood levels of α2-antiplasmin (a2AP), an ultrafast, covalent inhibitor of plasmin, have been linked in humans to increased risk of ischemic stroke and failure of tissue plasminogen activator (tPA) therapy. Consistent with these observations, a2AP neutralizes the therapeutic benefit of tPA therapy in experimental stroke. In addition, a2AP has deleterious, dose-related effects on ischemic brain injury in the absence of therapy. Experimental therapeutic inactivation of a2AP markedly reduces microvascular thrombosis, ischemic brain injury, brain swelling, brain hemorrhage, and death after thromboembolic stroke. These data provide new insights into the critical importance of a2AP in the pathogenesis of ischemic brain injury and suggest that transiently inactivating a2AP may have therapeutic value in ischemic stroke.

Microvascular thrombosis, fibrinolysis, ischemic injury, and death after cerebral thromboembolism are affected by levels of circulating α2-antiplasmin.

OBJECTIVE: Ischemic stroke is primarily attributable to thrombotic vascular occlusion. Elevated α2-antiplasmin (a2AP) levels correlate with increased stroke risk, but whether a2AP contributes to the pathogenesis of stroke is unknown. We examined how a2AP affects thrombosis, ischemic brain injury, and survival after experimental cerebral thromboembolism. APPROACH AND RESULTS: We evaluated the effects of a2AP on stroke outcomes in mice with increased, normal, or no circulating a2AP, as well as in mice given an a2AP-inactivating antibody. Higher a2AP levels were correlated with greater ischemic brain injury (rs=0.88, P<0.001), brain swelling (rs=0.82, P<0.001), and reduced middle cerebral artery thrombus dissolution (rs=-0.93, P<0.001). In contrast, a2AP deficiency enhanced thrombus dissolution, increased cerebral blood flow, reduced brain infarction, and decreased brain swelling. By comparison to tissue plasminogen activator (TPA), a2AP inactivation hours after thromboembolism still reduced brain infarction (P<0.001) and hemorrhage (P<0.05). Microvascular thrombosis, a process that enhances brain ischemia, was markedly reduced in a2AP-deficient or a2AP-inactivated mice compared with TPA-treated mice or mice with increased a2AP levels (all P<0.001). Matrix metalloproteinase-9 expression, which contributes to acute brain injury, was profoundly decreased in a2AP-deficient or a2AP-inactivated mice versus TPA-treated mice or mice with increased a2AP levels (all P<0.001). a2AP inactivation markedly reduced stroke mortality versus TPA (P<0.0001). CONCLUSIONS: a2AP has profound, dose-related effects on ischemic brain injury, swelling, hemorrhage, and survival after cerebral thromboembolism. By comparison to TPA, the protective effects of a2AP deficiency or inactivation seem to be mediated through reductions in microvascular thrombosis and matrix metalloproteinase-9 expression.

Signaling via P2Y12 may be critical for early stabilization of platelet aggregates.

P2Y(12) receptor antagonism inhibits platelet aggregation by preventing adenosine diphosphate (ADP)-mediated amplification of activation pathways downstream of primary agonists, such as thrombin and collagen. However, the role of ADP signaling in maintaining aggregate stability and the effects of P2Y(12) antagonists on preestablished aggregates in vitro and arterial thrombus in vivo are not well understood. This study evaluated the impact of P2Y(12) signaling on platelet aggregate stability and early thrombotic occlusion using a reversible P2Y(12) antagonist, ticagrelor. There were 2 study objectives: (1) to determine if there was a time-dependent factor on the capacity of a P2Y(12) antagonist to affect human platelet aggregate stability in vitro using light transmission aggregometry and (2) to evaluate the extent of arterial thrombus reversal in a preclinical model upon administration of ticagrelor in vivo. Platelet aggregates were exposed to ticagrelor after ADP or collagen activation, monitored for stability by aggregometry, and visualized by microscopy. Freshly formed ADP- and collagen-induced platelet aggregates were more rapidly dispersed by a P2Y(12) antagonist than drug carrier control at clinically relevant concentrations (P < 0.05). However, stable aggregates were not noticeably affected. A murine arterial thrombosis model was used to evaluate thrombus stability in an in vivo mouse model. Thrombotic occlusion was induced by FeCl(3), followed by a bolus intravenous administration of ticagrelor or vehicle control. Doppler blood flow was monitored before injury and 30 minutes after bolus administration. Arteries were retrieved for inspection for residual thrombus. Early arterial thrombotic occlusion in vivo was partially reversed by ticagrelor administration. Blood flow through the injured artery increased, and thrombus size within the artery decreased (P < 0.05, n = 3). In conclusion, P2Y(12) antagonism disrupts the stability of newly formed platelet aggregates, promoting disaggregation, and reverses thrombotic vascular occlusion. Thus, in addition to activating platelets, signaling via P2Y(12) seems to be required for stabilizing platelet thrombi.

Streptococcus uberis plasminogen activator (SUPA) activates human plasminogen through novel species-specific and fibrin-targeted mechanisms.

Bacterial plasminogen (Pg) activators generate plasmin to degrade fibrin blood clots and other proteins that modulate the pathogenesis of infection, yet despite strong homology between mammalian Pgs, the activity of bacterial Pg activators is thought to be restricted to the Pg of their host mammalian species. Thus, we found that Streptococcus uberis Pg activator (SUPA), isolated from a Streptococcus species that infects cows but not humans, robustly activated bovine but not human Pg in purified systems and in plasma. Consistent with this, SUPA formed a higher avidity complex (118-fold) with bovine Pg than with human Pg and non-proteolytically activated bovine but not human Pg. Surprisingly, however, the presence of human fibrin overrides the species-restricted action of SUPA. First, human fibrin enhanced the binding avidity of SUPA for human Pg by 4-8-fold in the presence and absence of chloride ion (a negative regulator). Second, although SUPA did not protect plasmin from inactivation by α(2)-antiplasmin, fibrin did protect human plasmin, which formed a 31-fold higher avidity complex with SUPA than Pg. Third, fibrin significantly enhanced Pg activation by reducing the K(m) (4-fold) and improving the catalytic efficiency of the SUPA complex (6-fold). Taken together, these data suggest that indirect molecular interactions may override the species-restricted activity of bacterial Pg activators; this may affect the pathogenesis of infections or may be exploited to facilitate the design of new blood clot-dissolving drugs.

Soluble (Pro)Renin Receptor Levels Are Regulated by Plasma Renin Activity and Correlated with Edema in Mice and Humans with HFrEF.

Symptomatic heart failure with reduced ejection fraction (HFrEF) is characterized by edema and chronic pathological activation of the classical renin-angiotensin-aldosterone system (RAAS). The soluble (pro)renin receptor (s(P)RR) is released into circulation by proteolytic cleavage of tissue expressed (P)RR and is a candidate biomarker of RAAS activation. However, previous studies linked elevated levels of s(P)RR in patients with HFrEF to renal dysfunction. Utilizing prospectively enrolled patients with comparable rEF, we show that increased plasma levels of s(P)RR are associated with symptomatic HF (characterized by edema), independent of chronic renal dysfunction. We also found that s(P)RR levels were positively correlated with patient plasma renin activity (PRA). Normotensive mice with dilated cardiomyopathy (DCM) and HFrEF, without renal dysfunction, showed plasma s(P)RR and PRA patterns similar to human HFrEF patients. Plasma s(P)RR levels positively correlated with PRA and systemic edema, but not with EF, resembling findings in patients with HFrEF without chronic kidney dysfunction. In female DCM mice with elevated PRA levels and plasma s(P)RR levels, a randomized, blinded trial comparing the direct renin inhibitor, aliskiren vs. vehicle control, showed that direct renin inhibition normalized PRA, lowered s(P)RR, and prevented symptomatic HFrEF. Considered in light of previous findings, these data suggest that, in HFrEF, in the absence of renal dysfunction, elevation of plasma s(P)RR levels is caused by increased PRA and associated with the development of systemic edema.

Sodium-Glucose Cotransporter-2 Inhibitors Improve Heart Failure with Reduced Ejection Fraction Outcomes by Reducing Edema and Congestion.

Pathological sodium-water retention or edema/congestion is a primary cause of heart failure (HF) decompensation, clinical symptoms, hospitalization, reduced quality of life, and premature mortality. Sodium-glucose cotransporter-2 inhibitors (SGLT-2i) based therapies reduce hospitalization due to HF, improve functional status, quality, and duration of life in patients with HF with reduced ejection fraction (HFrEF) independently of their glycemic status. The pathophysiologic mechanisms and molecular pathways responsible for the benefits of SGLT-2i in HFrEF remain inconclusive, but SGLT-2i may help HFrEF by normalizing salt-water homeostasis to prevent clinical edema/congestion. In HFrEF, edema and congestion are related to compromised cardiac function. Edema and congestion are further aggravated by renal and pulmonary abnormalities. Treatment of HFrEF patients with SGLT-2i enhances natriuresis/diuresis, improves cardiac function, and reduces natriuretic peptide plasma levels. In this review, we summarize current clinical research studies related to outcomes of SGLT-2i treatment in HFrEF with a specific focus on their contribution to relieving or preventing edema and congestion, slowing HF progression, and decreasing the rate of rehospitalization and cardiovascular mortality.

Suppression of Cardiogenic Edema with Sodium-Glucose Cotransporter-2 Inhibitors in Heart Failure with Reduced Ejection Fraction: Mechanisms and Insights from Pre-Clinical Studies.

In heart failure with reduced ejection fraction (HFrEF), cardiogenic edema develops from impaired cardiac function, pathological remodeling, chronic inflammation, endothelial dysfunction, neurohormonal activation, and altered nitric oxide-related pathways. Pre-clinical HFrEF studies have shown that treatment with sodium-glucose cotransporter-2 inhibitors (SGLT-2i) stimulates natriuretic and osmotic/diuretic effects, improves overall cardiac function, attenuates maladaptive cardiac remodeling, and reduces chronic inflammation, oxidative stress, and endothelial dysfunction. Here, we review the mechanisms and effects of SGLT-2i therapy on cardiogenic edema in various models of HFrEF. Overall, the data presented suggest a high translational importance of these studies, and pre-clinical studies show that SGLT-2i therapy has a marked effect on suppressing the progression of HFrEF through multiple mechanisms, including those that affect the development of cardiogenic edema.

Matrix Metalloproteinase-9 Expression is Enhanced by Ischemia and Tissue Plasminogen Activator and Induces Hemorrhage, Disability and Mortality in Experimental Stroke.

Matrix metalloproteinase-9 (MMP-9) degrades collagen and other cellular matrix proteins. After acute ischemic stroke, increased MMP-9 levels are correlated with hemorrhage, lack of reperfusion and stroke severity. Nevertheless, definitive data that MMP-9 itself causes poor outcomes in ischemic stroke are limited. In a model of experimental ischemic stroke with reperfusion, we examined whether ischemia and recombinant tissue plasminogen activator (r-tPA) therapy affected MMP-9 expression, and we used specific inhibitors to test if MMP-9 affects brain injury and recovery. After stroke, MMP-9 expression increased significantly in the ischemic vs. non-ischemic hemisphere of the brain (p < 0.001). MMP-9 expression in the ischemic, but not the non-ischemic hemisphere, was further increased by r-tPA treatment (p < 0.001). To determine whether MMP-9 expression contributed to stroke outcomes after r-tPA treatment, we tested three different antibody MMP-9 inhibitors. When compared to treatment with r-tPA and saline, treatment with r-tPA and MMP-9 antibody inhibitors significantly reduced brain hemorrhage by 11.3 to 38.6-fold (p < 0.01), brain swelling by 2.8 to 4.3-fold (p < 0.001) and brain infarction by 2.5 to 3.9-fold (p < 0.0001). Similarly, when compared to treatment with r-tPA and saline, treatment with r-tPA and an MMP-9 antibody inhibitor significantly improved neurobehavioral outcomes (p < 0.001), decreased weight loss (p < 0.001) and prolonged survival (p < 0.01). In summary, both prolonged ischemia and r-tPA selectively enhanced MMP-9 expression in the ischemic hemisphere. When administered with r-tPA, specific MMP-9 inhibitors markedly reduced brain hemorrhage, swelling, infarction, disability and death, which suggests that blocking the deleterious effects of MMP-9 may improve outcomes after ischemic stroke.

Platelet dense-granule secretion plays a critical role in thrombosis and subsequent vascular remodeling in atherosclerotic mice.

BACKGROUND: Platelet aggregation plays a critical role in myocardial infarction and stroke; however, the role of platelet secretion in atherosclerotic vascular disease is poorly understood. Therefore, we examined the hypothesis that platelet dense-granule secretion modulates thrombosis, inflammation, and atherosclerotic vascular remodeling after injury. METHODS AND RESULTS: Functional deletion of the Hermansky-Pudlak syndrome 3 gene (HPS3(-/-)) markedly reduces platelet dense-granule secretion. HPS3(-/-) mice have normal platelet counts, platelet morphology, and alpha-granule number, as well as maximal secretion of the alpha-granule marker P-selectin; however, their capacity to form platelet-leukocyte aggregates is significantly reduced (P<0.05). To examine the role of platelet dense-granule secretion in these processes, atherosclerosis-prone mice with combined genetic deficiency of apolipoprotein E and HPS3 (ApoE(-/-), HPS3(-/-)) were compared with congenic, atherosclerosis-prone mice with normal platelet secretion (ApoE(-/-), HPS3(+/+)). After 16 to 18 weeks on a high-fat diet, both groups of mice had similar fasting cholesterol levels and body weight. Carotid arteries of ApoE(-/-), HPS3(+/+) mice thrombosed rapidly after FeCl(3) injury, but ApoE(-/-), HPS3(-/-) mice were completely resistant to thrombotic arterial occlusion (P<0.01). Three weeks after injury, neointimal hyperplasia (from alpha-smooth muscle actin-positive cells) was significantly less (P<0.001) in arteries from ApoE(-/-), HPS3(-/-) mice. In ApoE(-/-), HPS3(-/-) mice, there were also pronounced reductions in arterial inflammation, as indicated by a 74% decrease in CD45-positive leukocytes (P<0.01) and a 73% decrease in Mac-3-positive macrophages (P<0.05). CONCLUSIONS: In atherosclerotic mice, reduced platelet dense-granule secretion is associated with marked protection against the development of arterial thrombosis, inflammation, and neointimal hyperplasia after vascular injury.