The 2019 coronavirus disease presents with a large variety of clinical manifestations ranging from asymptomatic carrier state to severe respiratory distress, multiple organ dysfunction and death. While it was initially considered primarily a respiratory illness, rapidly accumulating data suggests that COVID-19 results in a unique, profoundly prothrombotic milieu leading to both arterial and venous thrombosis. Consistently, elevated D-dimer level has emerged as an independent risk factor for poor outcomes, including death. Several other laboratory markers and blood counts have also been associated with poor prognosis, possibly due to their connection to thrombosis. At present, the pathophysiology underlying the hypercoagulable state is poorly understood. However, a growing body of data suggests that the initial events occur in the lung. A severe inflammatory response, originating in the alveoli, triggers a dysfunctional cascade of inflammatory thrombosis in the pulmonary vasculature, leading to a state of local coagulopathy. This is followed, in patients with more severe disease, by a generalized hypercoagulable state that results in macro-and microvascular thrombosis. Of concern, is the observation that anticoagulation may be inadequate in many circumstances, highlighting the need for alternative or additional therapies. Numerous ongoing studies investigating the pathophysiology of the COVID-19 associated coagulopathy may provide mechanistic insights that can direct appropriate interventional strategies.

The endothelium regulates vascular homeostasis, and endothelial dysfunction is a proximate event in the pathogenesis of atherothrombosis. Stimulation of the endothelium with proinflammatory cytokines or exposure to hemodynamic-induced disturbed flow leads to a proadhesive and prothrombotic phenotype that promotes atherothrombosis. In contrast, exposure to arterial laminar flow induces a gene program that confers a largely antiadhesive, antithrombotic effect. The molecular basis for this differential effect on endothelial function remains poorly understood. While recent insights implicate Kruppel-like factors (KLFs) as important regulators of vascular homeostasis, the in vivo role of these factors in endothelial biology remains unproven. Here, we show that endothelial KLF4 is an essential determinant of atherogenesis and thrombosis. Using in vivo EC-specific KLF4 overexpression and knockdown murine models, we found that KLF4 induced an antiadhesive, antithrombotic state. Mechanistically, we demonstrated that KLF4 differentially regulated pertinent endothelial targets via competition for the coactivator p300. These observations provide cogent evidence implicating endothelial KLFs as essential in vivo regulators of vascular function in the adult animal. IntroductionThrough the elaboration of numerous biological substances, ECs actively regulate fundamental physiological processes, such as regulation of blood coagulation, homing of immune cells, and barrier function. Studies over the past several decades have also identified key physiologic and pathologic phenotypic modulators of ECs. For example, stimulation of the endothelium with proinflammatory cytokines renders the endothelium dysfunctional, inducing a proadhesive and prothrombotic phenotype. In contrast, laminar flow induces critical genes that confer potent antithrombotic, antiadhesive, and antiinflammatory properties. The significance of fluid shear stress is evidenced by the observation that segments of the arterial tree exposed to laminar flow (e.g., straight regions of the vasculature) are less prone to the development of atherosclerotic lesions than are regions exposed to nonlaminar/disturbed flow (e.g., branch points). These observations have led to the current view that the balance of biochemical and biomechanical stimuli is the central determinant of vascular function under physiologic and pathologic conditions. Given the importance of the endothelium in vessel homeostasis, there is great interest in identifying molecular pathways that mediate the effects of both biochemical and biomechanical stimuli. Prior studies from our group and others have identified 2 members of the Kruppel-like factor (KLF) family of transcription factors, KLF2 and KLF4, as being of particular interest. Both KLF2 and KLF4 are induced by laminar flow and in in vitro stud-

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SUMMARY Precise control of myeloid cell activation is required for optimal host defense. However, this activation process must be under exquisite control to prevent uncontrolled inflammation. Herein, we identify the Kruppel-like transcription factor 2 (KLF2) as a potent regulator of myeloid cell activation in vivo. Exposure of myeloid cells to hypoxia and/or bacterial products reduced KLF2 expression while inducing hypoxia indusable factor-1α (HIF-1α), findings that were recapitulated in human septic patients. Myeloid KLF2 was found to be a potent inhibitor of nuclear factor-kappaB (NFκB)-dependent HIF-1α transcription and, consequently, a critical determinant of outcome in models of polymicrobial infection and endotoxemia. Collectively, these observations identify KLF2 as a tonic repressor of myeloid cell activation in vivo and an essential regulator of the innate immune system.

Despite significant evidence implicating an important role for neutrophils in thrombosis, their impact on the thrombotic process has remained a matter of controversy. Until 2010, platelets, coagulation factors, fibrinogen and monocytes were implicated in the thrombotic process. Several studies conducted over the last decade now support the growing notion that neutrophils indeed do contribute significantly to this process. Neutrophils can contribute to pathologic venous and arterial thrombosis or 'immunothrombosis' by the release of neutrophil extracellular traps (NETs) and NET release is emerging as a major contributor to thrombogenesis in pathologic situations such as sepsis and malignancy. Further, blood-cell derived microparticles, including those from neutrophils, have been implicated in thrombus formation. Finally, inflammasome activation in the neutrophil identifies another important mechanism that may be operative in neutrophil-driven risk for thrombosis. The knowledge of these roles of neutrophils in thrombosis may pave the road for novel anti-thrombotic agents in the future that do not affect hemostasis.

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Laminar shear stress is known to confer potent anti-inflammatory, antithrombotic, and antiadhesive effects by differentially regulating endothelial gene expression. The identification of Krüppel-like factor 2 as a flow-responsive molecule has greatly advanced our understanding of molecular mechanisms governing vascular homeostasis. This review summarizes the current understanding of Krüppel-like factor 2 action in endothelial gene expression and function.

Oligodeoxynucleotide libraries containing randomly incorporated bases are used to generate DNA aptamers by systematic evolution of ligands by exponential enrichment (SELEX). We predicted that combinatorial libraries with alternative base compositions might have innate properties different from the standard library containing equimolar A + C + G + T bases. In particular, we hypothesized that G-rich libraries would contain a higher proportion of quadruplex-forming sequences, which may impart desirable qualities, such as increased nuclease resistance and enhanced cellular uptake. Here, we report on 11 synthetic oligodeoxynucleotide libraries of various base combinations and lengths, with regard to their circular dichroism, stability in serum-containing medium, cellular uptake, protein binding and antiproliferative activity. Unexpectedly, we found that some G-rich libraries (composed of G + T or G + C nucleotides) strongly inhibited cancer cell growth while sparing non-malignant cells. These libraries had spectral features consistent with G-quadruplex formation, were significantly more stable in serum than inactive libraries and showed enhanced cellular uptake. Active libraries generally had strong protein binding, while the pattern of protein binding suggested that G/T and G/C libraries have distinct mechanisms of action. In conclusion, cancer-selective antiproliferative activity may be a general feature of certain G-rich oligodeoxynucleotides and is associated with quadruplex formation, nuclease resistance, efficient cellular uptake and protein binding.