Aggressive breast cancer in western Kenya has early onset, high proliferation, and immune cell infiltration.

BACKGROUND: Breast cancer incidence and mortality vary significantly among different nations and racial groups. African nations have the highest breast cancer mortality rates in the world, even though the incidence rates are below those of many nations. Differences in disease progression suggest that aggressive breast tumors may harbor a unique molecular signature to promote disease progression. However, few studies have investigated the pathology and clinical markers expressed in breast tissue from regional African patient populations. METHODS: We collected 68 malignant and 89 non-cancerous samples from Kenyan breast tissue. To characterize the tumors from these patients, we constructed tissue microarrays (TMAs) from these tissues. Sections from these TMAs were stained and analyzed using immunohistochemistry to detect clinical breast cancer markers, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor 2 receptor (HER2) status, Ki67, and immune cell markers. RESULTS: Thirty-three percent of the tumors were triple negative (ER-, PR-, HER2-), 59% were ER+, and almost all tumors analyzed were HER2-. Seven percent of the breast cancer patients were male, and 30% were <40 years old at diagnosis. Cancer tissue had increased immune cell infiltration with recruitment of CD163+ (M2 macrophage), CD25+ (regulatory T lymphocyte), and CD4+ (T helper) cells compared to non-cancer tissue. CONCLUSIONS: We identified clinical biomarkers that may assist in identifying therapy strategies for breast cancer patients in western Kenya. Estrogen receptor status in particular should lead initial treatment strategies in these breast cancer patients. Increased CD25 expression suggests a need for additional treatment strategies designed to overcome immune suppression by CD25+ cells in order to promote the antitumor activity of CD8+ cytotoxic T cells.

Unexpected role of anticoagulant protein C in controlling epithelial barrier integrity and intestinal inflammation.

The protein C (PC) pathway is a well-characterized coagulation system. Endothelial PC receptors and thrombomodulin mediate the conversion of PC to its activated form, a potent anticoagulant and anti-inflammatory molecule. Here we show that the PC pathway is expressed on intestinal epithelial cells. The epithelial expression of PC and endothelial PC receptor is down-regulated In patients with inflammatory bowel disease. PC(-/-)/PC(Tg) mice, expressing only 3% of WT PC, developed spontaneous intestinal inflammation and were prone to severe experimental colitis. These mice also demonstrated spontaneous elevated production of inflammatory cytokines and increased intestinal permeability. Structural analysis of epithelial tight junction molecules revealed that lack of PC leads to decreased JAM-A and claudin-3 expression and an altered pattern of ZO-1 expression. In vitro, treatment of epithelial cells with activated PC led to protection of tight junction disruption induced by TNF-α, and in vivo, topical treatment with activated PC led to mucosal healing and amelioration of colitis. Taken together, these findings demonstrate that the PC pathway is a unique system involved in controlling intestinal homeostasis and inflammation by regulating epithelial barrier function.

Plasminogen Deficiency Significantly Reduces Vascular Wall Disease in a Murine Model of Type IIa Hypercholesterolemia.

The fibrinolytic system has been implicated in the genesis and progression of atherosclerosis. It has been reported that a plasminogen (Pg) deficiency (Plg-/-) exacerbates the progression of atherosclerosis in Apoe-/- mice. However, the manner in which Plg functions in a low-density lipoprotein-cholesterol (LDL-C)-driven model has not been evaluated. To characterize the effect of Pg in an LDL-C-driven model, mice with a triple deficiency of the LDL-receptor (LDLr), along with the active component (apobec1) of the apolipoprotein B editosome complex, and Pg (L-/-/A-/-/Plg-/-), were generated. Atherosclerotic plaque formation was severely retarded in the absence of Pg. In vitro studies demonstrated that LDL uptake by macrophages was enhanced by plasmin (Pm), whereas circulating levels of LDL were enhanced, relative to L-/-/A-/- mice, and VLDL synthesis was suppressed. These results indicated that clearance of lipoproteins in the absence of LDLr may be regulated by Pg/Pm. Conclusions: The results from this study indicate that Pg exacerbates atherosclerosis in an LDL-C model of atherosclerosis and also plays a role in lipoprotein modification and clearance. Therefore, controlling the Pg system on macrophages to prevent foam cell formation would be a novel therapeutic approach.

A missense mutation in the sodium phosphate co-transporter Slc34a1 impairs phosphate homeostasis.

The sodium phosphate co-transporters Npt2a and Npt2c play important roles in the regulation of phosphate homeostasis. Slc34a1, the gene encoding Npt2a, resides downstream of the gene encoding coagulation factor XII (f12) and was inadvertently modified while generating f12(-/-) mice. In this report, the renal consequences of this modification are described. The combined single allelic mutant Slc34a1m contains two point mutations in exon 13: A499V is located in intracellular loop 5, and V528M is located in transmembrane domain 11. In addition to the expected coagulopathy of the f12(-/-) phenotype, mice homozygous for the double allelic modification (f12(-/-)/slc34a1(m/m)) displayed hypophosphatemia, hypercalcemia, elevated levels of alkaline phosphatase, urolithiasis, and hydronephrosis. Strategic cross-breedings demonstrated that the kidney-related pathology was associated only with autosomal recessive transmission of the slc34a1(m) gene and was not influenced by the simultaneous inactivation of f12. Npt2a[V528M] could be properly expressed in opossum kidney cells, but Npt2a[A499V] could not. These results suggest that a single amino acid substitution in Npt2a can lead to improper translocation of the protein to the cell membrane, disturbance of phosphate homeostasis, and renal calcification. Whether point mutations in the SLC34A1 gene can lead to hypophosphatemia and nephrolithiasis in humans remains unknown.

Plasminogen Activator Inhibitor-1 Protects Mice Against Cardiac Fibrosis by Inhibiting Urokinase-type Plasminogen Activator-mediated Plasminogen Activation.

Plasminogen activator inhibitor-1 (PAI-1) is known to protect mice against cardiac fibrosis. It has been speculated that PAI-1 may regulate cardiac fibrosis by inactivating urokinase-type plasminogen activator (uPA) and ultimately plasmin (Pm) generation. However, the in vivo role of PAI-1 in inactivating uPA and limiting the generation of Pm during cardiac fibrosis remains to be established. The objective of this study was to determine if the cardioprotective effect of PAI-1 is mediated through its ability to directly regulate urokinase -mediated activation of plasminogen (Pg). An Angiotensin II (AngII)-aldosterone (Ald) infusion mouse model of hypertension was utilised in this study. Four weeks after AngII-Ald infusion, PAI-1-deficient (PAI-1-/-) mice developed severe cardiac fibrosis. However, a marked reduction in cardiac fibrosis was observed in PAI-1-/-/uPA-/- double knockout mice that was associated with reduced inflammation, lower expression levels of TGF-ß and proteases associated with tissue remodeling, and diminished Smad2 signaling. Moreover, total ablation of cardiac fibrosis was observed in PAI-1-/- mice that express inactive plasmin (Pm) but normal levels of zymogen Pg (PAI-1-/-/PgS743A/S743A). Our findings indicate that PAI-1 protects mice from hypertension-induced cardiac fibrosis by inhibiting the generation of active Pm.

Tumor infiltrating leukocyte density is independent of tumor grade and molecular subtype in aggressive breast cancer of Western Kenya.

BACKGROUND: Tumors commonly are infiltrated by leukocytes, or tumor infiltrating leukocytes (TILs). It remains unclear, however, if the density and type of individual TILs has a direct or simply correlative role in promoting poor prognosis in breast cancer patients. Breast cancer in Kenyan women is aggressive with presentation at a young age, with advanced grade (grade III), large tumor size (>2.0 cm), and poor prognosis. We previously observed that the tumors were predominantly estrogen receptor positive (ER+) but also included both a high percentage of triple negative tumors and also increased immune cell infiltration within the tumors. We used breast tumor tissues from each patient to make tissue microarrays that were then stained for leukocyte and myeloid markers including CD4, CD8, CD20, CD25, CD68, and CD163 using immunohistochemical techniques. The immune cell infiltration into the cancer tissue included increased numbers of macrophages (CD68+), helper T cells (CD4+), and CD25+ lymphocytes compared to benign tissue. RESULTS: This study characterized the grade, molecular subtypes, and proliferation index of these tumors and determined if TIL density was enriched across any of these factors. We analyzed 49 malignant patient tissue samples for this study. The patient population had a mean age of 51.9 years. The tumors analyzed were heterogeneous by grade: grade I (6%), grade II (47%), and grade III (39%). Most patients presented with large tumors (>2.0 cm) (69%). We classified the tumors into molecular subtypes based on clinical marker expression. Based on this analysis, the molecular subtype distribution was heterogeneous with luminal B (41%), basal/triple negative (TN) (37%), luminal A (14%) and HER2 (8%) breast cancer subtypes. While the basal/TN subtype had a much higher proliferative index (Ki-67+) than did the other molecular subtypes, we did not see a significant correlation between TIL density and either subtype or tumor grade. Therefore, TIL density is independent of molecular subtype and grade. CONCLUSION: This study identified a Kenyan patient cohort that develops large, high-grade tumors primarily of the luminal B and basal molecular subtypes. After analyzing the TILs within these tumors, we found that immune cell infiltration of these tumors correlated with increased proliferation but not grade or molecular subtype. Future research is required to determine if the aberrant recruitment of TILs to tumors contributes to cancer progression and response to cancer treatments.

Focal arterial inflammation is augmented in mice with a deficiency of the protein C gene.

Increased risk of thrombosis, with propitious conditions for fibrin deposition, along with upregulation of inflammation, are important factors that enhance plaque formation in atherosclerosis. Evidence supporting the role of anticoagulant protein C (PC) as an inflammatory agent has emerged, supplementing its well-known function as an anticoagulant. Thus, we sought to examine whether a PC deficiency would lead to an enhanced response to an acute arterial hyperplasic challenge. The presentation of early arterial inflammation was studied using a copper/silicone arterial cuff model of accelerated focal neointimal remodeling in mice with a heterozygous total deficiency of PC (PC+/-). Increased inflammation, cell proliferation, cell migration, fibrin elevation, and tissue necrosis were observed in the treated arteries of PC+/- mice, as compared to arteries of equally challenged age- and gender-matched WT mice. These results indicate that PC+/- mice subjected to this challenge displayed enhanced focal arterial inflammation and thrombosis, leading to larger neointimas and subsequent localized occlusion, as compared to their WT counterparts.

Abrogation of plasminogen activator inhibitor-1-vitronectin interaction ameliorates acute kidney injury in murine endotoxemia.

Sepsis-induced acute kidney injury (AKI) contributes to the high mortality and morbidity in patients. Although the pathogenesis of AKI during sepsis is poorly understood, it is well accepted that plasminogen activator inhibitor-1 (PAI-1) and vitronectin (Vn) are involved in AKI. However, the functional cooperation between PAI-1 and Vn in septic AKI has not been completely elucidated. To address this issue, mice were utilized lacking either PAI-1 (PAI-1-/-) or expressing a PAI-1-mutant (PAI-1R101A/Q123K) in which the interaction between PAI-1 and Vn is abrogated, while other functions of PAI-1 are retained. It was found that both PAI-1-/- and PAI-1R101A/Q123K mice are associated with decreased renal dysfunction, apoptosis, inflammation, and ERK activation as compared to wild-type (WT) mice after LPS challenge. Also, PAI-1-/- mice showed attenuated fibrin deposition in the kidneys. Furthermore, a lack of PAI-1 or PAI-1-Vn interaction was found to be associated with an increase in activated Protein C (aPC) in plasma. These results demonstrate that PAI-1, through its interaction with Vn, exerts multiple deleterious mechanisms to induce AKI. Therefore, targeting of the PAI-1-Vn interaction in kidney represents an appealing therapeutic strategy for the treatment of septic AKI by not only altering the fibrinolytic capacity but also regulating PC activity.

Mice with a severe deficiency of the endothelial protein C receptor gene develop, survive, and reproduce normally, and do not present with enhanced arterial thrombosis after challenge.

The endothelial cell Protein C receptor (EPCR) functions to enhance activation of anticoagulant Protein C (PC) by the thrombin/ thrombomodulin (Tm) complex on the surface of the endothelium. This overall system functions in anticoagulation, profibrinolytic, and antiinflammatory responses. Mice with a severe targeted deficiency of this receptor have been generated by integration of exogenous DNA elements into the 5'-untranslated region of the EPCR gene. Despite the retention of the entire endogenous EPCR coding sequence in the altered EPCR gene locus, only very low EPCR message contents were detected in mice by quantitative RT-PCR during embryogenesis and up to at least early adulthood. Immunohistochemical analysis of various regions of the arterial tree of mice up to 4 months of age, employing an anti-murine EPCR antibody, confirmed that undetectable levels of this protein were present in arterial regions during these periods. Despite this, these mice are not more prone to arterial thrombosis after challenge in a FeCl3 carotid artery thrombosis model. Small amounts (<10% of wild-type) of this protein were found in other tissues. Matings of mice homozygous for this deficiency led to normal births and survival of the offspring, in contrast to results by others demonstrating early embryonic lethality of a total EPCR deficiency. These data further show that minimal levels of EPCR are able to support male and female virility, as well as embryonic development, birth, and survival to adulthood.

Systemic platelet dysfunction is the result of local dysregulated coagulation and platelet activation in the brain in a rat model of isolated traumatic brain injury.

Coagulopathy after severe traumatic brain injury (TBI) has been extensively reported. Clinical studies have identified a strong relationship between diminished platelet-rich thrombus formation, responsiveness to adenosine diphosphate agonism, and severity of TBI. The mechanisms that lead to platelet dysfunction in the acute response to TBI are poorly understood. The development of a rodent model of TBI that mimics the coagulopathy observed clinically has recently been reported. Using immunohistochemical techniques and thromboelastography platelet mapping, the current study demonstrated that the expression of coagulation (tissue factor and fibrin) and platelet activation (P-selectin) markers in the injured brain paralleled the alteration in systemic platelet responsiveness to the agonists, adenosine diphosphate and arachodonic acid. Results of this study demonstrate that local procoagulant changes in the injured brain have profound effects on systemic platelet function.

Early platelet dysfunction in a rodent model of blunt traumatic brain injury reflects the acute traumatic coagulopathy found in humans.

Acute coagulopathy is a serious complication of traumatic brain injury (TBI) and is of uncertain etiology because of the complex nature of TBI. However, recent work has shown a correlation between mortality and abnormal hemostasis resulting from early platelet dysfunction. The aim of the current study was to develop and characterize a rodent model of TBI that mimics the human coagulopathic condition so that mechanisms of the early acute coagulopathy in TBI can be more readily assessed. Studies utilizing a highly reproducible constrained blunt-force brain injury in rats demonstrate a strong correlation with important postinjury pathological changes that are observed in human TBI patients, namely, diminished platelet responses to agonists, especially adenosine diphosphate (ADP), and subarachnoid bleeding. Additionally, administration of a direct thrombin inhibitor, preinjury, recovers platelet functionality to ADP stimulation, indicating a direct role for excess thrombin production in TBI-induced early platelet dysfunction.

Abnormal whole blood thrombi in humans with inherited platelet receptor defects.

To delineate the critical features of platelets required for formation and stability of thrombi, thromboelastography and platelet aggregation measurements were employed on whole blood of normal patients and of those with Bernard-Soulier Syndrome (BSS) and Glanzmann's Thrombasthenia (GT). We found that separation of platelet activation, as assessed by platelet aggregation, from that needed to form viscoelastic stable whole blood thrombi, occurred. In normal human blood, ristocetin and collagen aggregated platelets, but did not induce strong viscoelastic thrombi. However, ADP, arachidonic acid, thrombin, and protease-activated-receptor-1 and -4 agonists, stimulated both processes. During this study, we identified the genetic basis of a very rare double heterozygous GP1b deficiency in a BSS patient, along with a new homozygous GP1b inactivating mutation in another BSS patient. In BSS whole blood, ADP responsiveness, as measured by thrombus strength, was diminished, while ADP-induced platelet aggregation was normal. Further, the platelets of 3 additional GT patients showed very weak whole blood platelet aggregation toward the above agonists and provided whole blood thrombi of very low viscoelastic strength. These results indicate that measurements of platelet counts and platelet aggregability do not necessarily correlate with generation of stable thrombi, a potentially significant feature in patient clinical outcomes.

A fibrinogen deficiency accelerates the initiation of LDL cholesterol-driven atherosclerosis via thrombin generation and platelet activation in genetically predisposed mice.

Mice with combined deficiencies of the low-density lipoprotein receptor (LDLR(-/-)) and the catalytic component of an apolipoprotein B-edisome complex (APOBEC1(-/-)) that converts apoB-100 to apoB-48 have been characterized, and this model of LDL cholesterol-driven atherosclerosis was applied to an investigation of the role of fibrinogen (Fg) in the genesis and progression of the plaque. LDLR(-/-)/APOBEC1(-/-)/FG(-/-) (L(-/-)/A(-/-)/FG(-/-)) triple-deficient mice presented more advanced plaque in their aortic trees and aortic sinuses at 24, 36, and 48 weeks of age compared to L(-/-)/A(-/-) mice, a feature that may result from enhanced platelet activation in these former mice. This is supported by the presence of hypercoagulability, increased CD61 and CD62P on resting platelets, and higher plasma soluble P-selectin in L(-/-)/A(-/-)/FG(-/-) mice as compared to L(-/-)/A(-/-), FG(-/-), or wild-type mice. The elevated higher molecular weight forms of von Willebrand factor (VWF) in L(-/-)/A(-/-)/FG(-/-) mice, revealed by increased VWF collagen binding activity, perhaps resulting from down-regulation of its cleaving metalloproteinase, ADAMTS13, further indicates enhanced platelet activation. Thus, the earlier arterial plaque deposition in L(-/-)/A(-/-)/FG(-/-) mice appears to contain a contribution from enhanced levels of thrombin and activated platelets, a synergistic consequence of an Fg deficiency combined with a high LDL cholesterol concentration.

Fibrinogen stabilizes placental-maternal attachment during embryonic development in the mouse.

In humans, maternal fibrinogen (Fg) is required to support pregnancies by maintaining hemostatic balance and stabilizing uteroplacental attachment at the fibrinoid layer found at the fetal-maternal junction. To examine relationships between low Fg levels and early fetal loss, a genetic model of afibrinogenemia was developed. Pregnant mice homozygous for a deletion of the Fg-gamma chain, which results in a total Fg deficiency state (FG(-/-)), aborted the fetuses at the equivalent gestational stage seen in humans. Results obtained from timed matings of FG(-/-) mice showed that vaginal bleeding was initiated as early as embryonic day (E)6 to 7, a critical stage for maternal-fetal vascular development. The condition of afibrinogenemia retarded embryo-placental development, and consistently led to abortion and maternal death at E9.75. Lack of Fg did not alter the extent or distribution pattern of other putative factors of embryo-placental attachment, including laminin, fibronectin, and Factor XIII, indicating that the presence of fibrin(ogen) is required to confer sufficient stability at the placental-decidual interface. The results of these studies demonstrate that maternal Fg plays a critical role in maintenance of pregnancy in mice, both by supporting proper development of fetal-maternal vascular communication and stabilization of embryo implantation.

Enhanced in vitro proliferation of aortic endothelial cells from plasminogen activator inhibitor-1-deficient mice.

A number of studies have identified a role for plasminogen activator inhibitor-1 (PAI-1) in regulating angiogenesis, although results from these investigations have been controversial. Among key cellular components of an angiogenic vessel are endothelial cells (ECs), which are known to express several components of the fibrinolytic system, including PAI-1. Thus, alterations in expression of this protein may have direct effects on cell functions involved in vascular development. In this study, ECs were isolated from sections of murine arterial trees from wild-type and PAI-1-deficient mice, and low passage (passages 3-4) homogeneous subpopulations of these cells were obtained by immunomagnetic absorption to antibodies against CD105/CD106. The homogeneity of these cells was further assessed by immunohistochemistry and quantitative real-time reverse transcription-PCR analysis of a number of EC markers. Comparative analyses of EC proliferation (one event associated with angiogenesis) in wild-type and PAI-1-deficient ECs demonstrated enhanced rates of cell growth for PAI-1-deficient cells relative to wild-type cells. Additional studies demonstrated similar levels of both vascular endothelial growth factor (VEGF) mRNA and protein and enhanced levels of VEGF receptor-1 (Flt-1) mRNA in PAI-1-deficient cells relative to wild-type cells. Immunohistochemical analyses indicated that phosphorylation of Akt was also enhanced in PAI-1-deficient cells, implicating VEGF-induced cell signaling alterations in PAI-1-deficient cells, the result of which may contribute to alterations in cell proliferation.