Stem Cell Therapy for Wound Healing in Ischemic Limbs: Is It Effective?

Critical limb ischemia is an important clinical entity due to its association with increased morbidity and mortality. The mortality and amputation-free survival remains poor especially in those where revascularization is not an option. Recently, the role of cellular therapy has emerged as a promising therapeutic measure that may aid in wound healing and revascularization and improve functional outcomes.

Transfection of hypoxia-inducible factor-1α mRNA upregulates the expression of genes encoding angiogenic growth factors.

Hypoxia-Inducible Factor-1α (HIF-1α) has presented a new direction for ischemic preconditioning of surgical flaps to promote their survival. In a previous study, we demonstrated the effectiveness of HIF-1a DNA plasmids in this application. In this study, to avoid complications associated with plasmid use, we sought to express HIF-1α through mRNA transfection and determine its biological activity by measuring the upregulation of downstream angiogenic genes. We transfected six different HIF-1a mRNAs-one predominant, three variant, and two novel mutant isoforms-into primary human dermal fibroblasts using Lipofectamine, and assessed mRNA levels using RT-qPCR. At all time points examined after transfection (3, 6, and 10 h), the levels of HIF-1α transcript were significantly higher in all HIF-1α transfected cells relative to the control (all p < 0.05, unpaired Student's T-test). Importantly, the expression of HIF-1α transcription response genes (VEGF, ANG-1, PGF, FLT1, and EDN1) was significantly higher in the cells transfected with all isoforms than with the control at six and/or ten hours post-transfection. All isoforms were transfected successfully into human fibroblast cells, resulting in the rapid upregulation of all five downstream angiogenic targets tested. These findings support the potential use of HIF-1α mRNA for protecting ischemic dermal flaps.

Mesenchymal Stem Cell Culture within Perfusion Bioreactors Incorporating 3D-Printed Scaffolds Enables Improved Extracellular Vesicle Yield with Preserved Bioactivity.

Extracellular vesicles (EVs) are implicated as promising therapeutics and drug delivery vehicles in various diseases. However, successful clinical translation will depend on the development of scalable biomanufacturing approaches, especially due to the documented low levels of intrinsic EV-associated cargo that may necessitate repeated doses to achieve clinical benefit in certain applications. Thus, here the effects of a 3D-printed scaffold-perfusion bioreactor system are assessed on the production and bioactivity of EVs secreted from bone marrow-derived mesenchymal stem cells (MSCs), a cell type widely implicated in generating EVs with therapeutic potential. The results indicate that perfusion bioreactor culture induces an ≈40-80-fold increase (depending on measurement method) in MSC EV production compared to conventional cell culture. Additionally, MSC EVs generated using the perfusion bioreactor system significantly improve wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Overall, this study establishes a promising solution to a major EV translational bottleneck, with the capacity for tunability for specific applications and general improvement alongside advancements in 3D-printing technologies.

Impact of storage conditions and duration on function of native and cargo-loaded mesenchymal stromal cell extracellular vesicles.

BACKGROUND AIMS: As evidenced by ongoing clinical trials and increased activity in the commercial sector, extracellular vesicle (EV)-based therapies have begun the transition from bench to bedside. As this progression continues, one critical aspect of EV clinical translation is understanding the effects of storage and transport conditions. Several studies have assessed the impact of storage on EV characteristics such as morphology, uptake and component content, but effects of storage duration and temperature on EV functional bioactivity and, especially, loaded cargo are rarely reported. METHODS: The authors assessed EV outcomes following storage at different temperatures (room temperature, 4°C, -20°C, -80°C) for various durations as well as after lyophilization. RESULTS: Mesenchymal stromal cell (MSC) EVs were observed to retain key aspects of their bioactivity (pro-vascularization, anti-inflammation) for up to 4-6 weeks at -20°C and -80°C and after lyophilization. Furthermore, via in vitro assays and an in vivo wound healing model, these same storage conditions were also demonstrated to enable preservation of the functionality of loaded microRNA and long non-coding RNA cargo in MSC EVs. CONCLUSIONS: These findings extend the current understanding of how EV therapeutic potential is impacted by storage conditions and may inform best practices for handling and storing MSC EVs for both basic research and translational purposes.

Pioneering use of genetic analysis for CDH1 to identify candidates for prophylactic total gastrectomy to prevent hereditary diffuse gastric cancer.

Worldwide, gastric cancer results in significant morbidity and mortality. Ten per cent of patients with gastric cancer have a strong family history of the disease. CDH1 (E-cadherin) has been identified as a key gene whose mutation leads to hereditary diffuse gastric cancer. We overviewed 33 articles with prophylactic total gastrectomy and assessed the outcomes and benefits. Families with mutations in CDH1 may benefit from early prophylactic total gastrectomy. Dr Mark Duncan has applied his experience as a high-volume gastric cancer surgeon to treat not only individual patients, but several generations of patients within a family. This use of prophylactic total gastrectomy is well tolerated by patients and prevents the future development of gastric cancer.

Potential Role of Silencing Ribonucleic Acid for Esophageal Cancer Treatment.

INTRODUCTION: Esophageal cancer is an aggressive malignancy with high mortality. Optimal treatment of esophageal cancer remains an elusive goal. Ribonucleic acid (RNA) interference is a novel potential targeted approach to treat esophageal cancer. Targeting oncogenes that can alter critical cellular functions with silencing RNA molecules is a promising approach. The silencing of specific oncogenes in esophageal cancer cells in the experimental setting has been shown to decrease the expression of oncogenic proteins. This has resulted in cell apoptosis, reduction in cell proliferation, reduced invasion, migration, epithelial-mesenchymal transition, decrease in tumor angiogenesis and metastasis, and overcoming drug resistance. The Hedgehog (Hh) signaling pathway has been shown to be involved in esophageal adenocarcinoma formation in a reflux animal model. In addition to Hh, we will focus on other targets with clinical potential in the treatment of esophageal cancer. MATERIALS AND METHODS: We searched for articles published from 2005 to August 2020 that studied the siRNA effects on inhibiting esophageal cancer formation in experimental settings. We used combinations of the following terms for searching: "esophageal cancer," "RNA interference," "small interfering RNA," "siRNA," "silencing RNA," "Smoothened (Smo)," "Gli," "Bcl-2," "Bcl-XL," "Bcl-W,″ "Mcl-1," "Bfl-1," "STAT3,"and "Hypoxia inducible factor (HIF)". A total of 21 relevant articles were found. RESULTS AND CONCLUSIONS: Several proto-oncogenes/oncogenes including Hh pathway mediators, glioma-associated oncogene homolog 1 (Gli-1), Smoothened (Smo), and antiapoptotic Bcl-2 have potential as targets for silencing RNA in the treatment of esophageal cancer.

HOTAIR-Loaded Mesenchymal Stem/Stromal Cell Extracellular Vesicles Enhance Angiogenesis and Wound Healing.

Chronic wounds remain a substantial source of morbidity worldwide. An emergent approach that may be well-suited to induce the complex, multicellular processes such as angiogenesis that are required for wound repair is the use of extracellular vesicles (EVs). EVs contain a wide variety of proteins and nucleic acids that enable multifactorial signaling. Here, the capability of EVs is leveraged to be engineered via producer cell modification to investigate the therapeutic potential of EVs from mesenchymal stem/stromal cells (MSCs) transfected to overexpress long non-coding RNA HOX transcript antisense RNA (HOTAIR). HOTAIR is previously shown by the authors' group to be critical in mediating angiogenic effects of endothelial cell EVs, and MSCs are chosen as EV producer cells for this study due to their widely reported intrinsic angiogenic properties. The results indicate that MSCs overexpressing HOTAIR (HOTAIR-MSCs) produce EVs with increased HOTAIR content that promote angiogenesis and wound healing in diabetic (db/db) mice. Further, endothelial cells exposed to HOTAIR-MSC EVs exhibit increased HOTAIR content correlated with upregulation of the angiogenic protein vascular endothelial growth factor. Thus, this study supports EV-mediated HOTAIR delivery as a strategy for further exploration toward healing of chronic wounds.

Targeting the Hedgehog Pathway Using Itraconazole to Prevent Progression of Barrett's Esophagus to Invasive Esophageal Adenocarcinoma.

OBJECTIVE: The aim of the study was to investigate whether inhibition of Sonic Hedgehog (SHH) pathway would prevent progression of Barrett's Esophagus (BE) to esophageal adenocarcinoma. BACKGROUND: The hedgehog signaling pathway is a leading candidate as a molecular mediator of BE and esophageal adenocarcinoma (EAC). Repurposed use of existing off-patent, safe and tolerable drugs that can inhibit hedgehog, such as itraconazole, could prevent progression of BE to EAC. METHODS: The efficacy of itraconazole was investigated using a surgical rat reflux model of Barrett's Metaplasia (BM). Weekly intraperitoneal injections of saline (control group) or itraconazole (treatment group; 200 mg/kg) were started at 24 weeks postsurgery. Esophageal tissue was harvested at 40 weeks. The role of the Hh pathway was also evaluated clinically. Esophageal tissue was harvested after 40 weeks for pathological examination and evaluation of the SHH pathway by immunohistochemistry. RESULTS: BM was present in control animals 29 of 31 (93%) versus itraconazole 22 of 24 (91%). EAC was significantly lower in itraconazole 2 of 24 (8%) versus control 10 of 31 (32%), respectively (P = 0.033). Esophageal SHH levels were lower in itraconazole vs control (P = 0.12). In esophageal tissue from humans with recurrent or persistent dysplastic BE within 24 months of ablative treatment, strong SHH and Indian Hedgehog expression occurred in distal BE versus proximal squamous epithelium, odds ratio = 6.1 (95% confidence interval: 1.6, 23.4) and odds ratio = 6.4 (95% confidence interval: 1.2, 32.8), respectively. CONCLUSION: Itraconazole significantly decreases EAC development and SHH expression in a preclinical animal model of BM. In humans, BE tissue expresses higher SHH, Indian Hedgehog, and bone morphogenic protein levels than normal squamous esophageal epithelium.

Preconditioning of surgical pedicle flaps with DNA plasmid expressing hypoxia-inducible factor-1α (HIF-1α) promotes tissue viability.

Ischemic necrosis of surgical flaps after reconstruction is a major clinical problem. Hypoxia-inducible factor-1α (HIF-1α) is considered the master regulator of the adaptive response to hypoxia. Among its many properties, it regulates the expression of genes encoding angiogenic growth factors, which have a short half-life in vivo. To achieve a continuous application of the therapeutic, we utilized DNA plasmid delivery. Transcription of the DNA plasmid confirmed by qRT-PCR showed significantly increased mRNA for HIF-1α in the transfected tissue compared to saline control tissue. Rats were preconditioned by injecting with either HIF-1α DNA plasmid or saline intradermally in the designated flap region on each flank. Seven days after preconditioning, each rat had two isolated pedicle flaps raised with a sterile silicone sheet implanted between the skin flap and muscle layer. The flaps preconditioned with HIF-1α DNA plasmid had significantly less necrotic area. Angiogenesis measured by CD31 staining showed a significant increase in the number of vessels per high powered field in the HIF-1α group (p < 0.05). Our findings offer a potential therapeutic strategy for significantly promoting the viability of surgical pedicle flaps by ischemic preconditioning with HIF-1α DNA plasmid.

Therapeutic potential of extracellular vesicle-associated long noncoding RNA.

Both extracellular vesicles (EVs) and long noncoding RNAs (lncRNAs) have been increasingly investigated as biomarkers, pathophysiological mediators, and potential therapeutics. While these two entities have often been studied separately, there are increasing reports of EV-associated lncRNA activity in processes such as oncogenesis as well as tissue repair and regeneration. Given the powerful nature and emerging translational impact of other noncoding RNAs such as microRNA (miRNA) and small interfering RNA, lncRNA therapeutics may represent a new frontier. While EVs are natural vehicles that transport and protect lncRNAs physiologically, they can also be engineered for enhanced cargo loading and therapeutic properties. In this review, we will summarize the activity of lncRNAs relevant to both tissue repair and cancer treatment and discuss the role of EVs in enabling the potential of lncRNA therapeutics.

Total Gastrectomy for CDH-1 Mutation Carriers: An Institutional Experience.

BACKGROUND: Gastric cancer is a leading cause of cancer-related death across the world. A subset of gastric cancers demonstrates an inherited genetic predisposition. Individuals with germline mutations in the CDH1 gene incur a lifetime risk for diffuse gastric cancer and benefit from prophylactic gastrectomy. The results for this operative intervention remain relatively undescribed in the literature, despite guidelines supporting its use. METHODS: We present a single-institution series of patients with confirmed CDH1 mutations who underwent gastrectomy. We describe their presenting symptoms, preoperative screening, clinicopathologic features, and outcomes. Focal outcomes of interest are weight loss and postoperative morbidity. RESULTS: Between 2010 and 2018, ten patients with a confirmed CDH1 mutation underwent total gastrectomy with intestinal pouch reconstruction at our institution. Two patients had clinical gastric cancer at the time of their operation at 21 and 60 y of age. Eight patients had prophylactic gastrectomy. All prophylactic patients had undergone prior endoscopic screening without detection of cancer; however, three had occult gastric cancer on pathological examination. Median weight loss after gastrectomy was 10 kg at 6 mo and 11 kg at 1 y. Postoperative morbidity was limited to one anastomotic leak, one hematoma, and one case of pneumonia. All patients remain disease-free with median follow-up of 19 mo. CONCLUSIONS: Total gastrectomy for patients with a CDH1 mutation is a cancer-preventing operation for a high-risk population. For this series, jejunal pouch reconstruction was performed with encouragingly low postoperative morbidity, weight loss, and good subjective function.

Impact of Extravasated Platelet Activation and Podoplanin-positive Cancer-associated Fibroblasts in Pancreatic Cancer Stroma.

BACKGROUND/AIM: The aim of the study was to evaluate the status of extravasated platelet activation (EPA) surrounding podoplanin (PDPN)-positive cancer-associated fibroblasts (CAFs) in pancreatic cancer stroma by neoadjuvant chemotherapy. PATIENTS AND METHODS: A total of 74 patients were enrolled in this study. We investigated CD42b and PDPN expression in the groups of untreated, gemcitabine (GEM) alone, GEM plus S-1 (GS) and GEM plus nab-paclitaxel (GnP). RESULTS: CD42b expression in surrounding CAFs was observed in 58% patients. CD42b expression was significantly correlated with PDPN expression. CD42b-positive cases were significantly lower in the group treated with GnP than in the untreated group and groups treated with GEM alone or GS. PDPN expression was reduced in the GnP group, as revealed by markedly disorganized collagen and a low density of PDPN-positive fibroblasts. There was a significantly lower CD42b expression and fewer PDPN-positive fibroblasts in the GnP group than in untreated, GEM alone, and GS groups, but there was no significant difference between the latter three groups. CONCLUSION: There is a significant association between EPA and PDPN-positive CAFs in pancreatic cancer stroma. Our data suggest that the GnP regimen decreases EPA through PDPN-positive CAF depletion.

Krüppel-like Factor 5 Promotes Sonic Hedgehog Signaling and Neoplasia in Barrett's Esophagus and Esophageal Adenocarcinoma.

Krüppel-like Factor 5 (KLF5) is a zinc-finger transcription factor associated with cell cycle progression and cell survival. KLF5 plays a key role in mammalian intestinal epithelium development and maintenance, expressed at high levels in basal proliferating cells and low levels in terminally differentiated cells. Considering Barrett's esophagus (BE) and esophageal adenocarcinoma's (EAC) histopathological similarities to intestinal epithelium, we sought to determine KLF5's role in BE and EAC, as well as KLF5's possible connection to the sonic hedgehog (SHH) pathway which is highly active in BE and EAC development. Low levels of KLF5 mRNA were found in BE cell lines and tissue- similar to what has been reported in differentiated intestinal epithelium. In contrast, higher KLF5 levels were observed in EAC cells and tissues. KLF5 knockdown in EAC cells caused significant decreases in cell migration, proliferation, and EAC-associated gene expression. Moreover, KLF5 knockdown led to decreased SHH signaling. These results suggest that KLF5 is connected to the SHH pathway in BE and EAC and may represent a potential drug target in EAC; further studies are now indicated to verify these findings and elucidate underlying mechanisms involved.

Engineering Pro-Regenerative Hydrogels for Scarless Wound Healing.

Skin and skin appendages protect the body from harmful environment and prevent internal organs from dehydration. Superficial epidermal wounds usually heal without scarring, however, deep dermal wound healing commonly ends up with nonfunctioning scar formation with substantial loss of skin appendage. Wound healing is one of the most complex dynamic biological processes, during which a cascade of biomolecules combine with stem cell influx and matrix synthesis and synergistically contribute to wound healing at all levels. Although many approaches have been investigated to restore complete skin, the clinically effective therapy is still unavailable and the regeneration of perfect skin still remains a significant challenge. The complete mechanism behind scarless skin regeneration still requires further investigation. Fortunately, recent advancement in regenerative medicine empowers us more than ever to restore tissue in a regenerative manner. Many studies have elucidated and reviewed the contribution of stem cells and growth factors to scarless wound healing. This article focuses on recent advances in scarless wound healing, especially strategies to engineer pro-regenerative scaffolds to restore damaged skin in a regenerative manner.

Preventive effect of oral hangeshashinto (TJ-14) on the development of reflux-induced esophageal cancer.

BACKGROUND: Prostaglandin E2 is one of the potential products that promotes development of tumors and also is a strong inducer of M2 phenotype macrophages, which contribute to tumor development in the immunosuppressed microenvironment. Hangeshashinto (TJ-14), a Japanese traditional medicine (Kampo medicine), has been reported to be effective in preventing chemotherapy-induced oral mucositis through the reduction of prostaglandin E2. We previously developed a surgical rat reflux model of esophageal cancer and used this well-established animal model to investigate the action of TJ-14 in preventing esophageal cancer. We also assessed the effect of TJ-14 on the downregulation of prostaglandin E2 production, utilizing esophageal squamous cell carcinoma cell line exposed to bile acid. METHODS: An end-to-side esophagojejunostomy was performed for the reflux model. A daily oral diet was subsequently administered, consisting of either diet-incorporated TJ-14 or standard diet as a control group. The rats were killed at 40 weeks after surgery. The incidence of esophageal cancer, Barrett's metaplasia, and proliferative hyperplasia were assessed histologically. CD163, a M2 phenotype macrophage marker, was assessed with immunohistochemistry. Prostaglandin E2 enzyme immunoassay and lactate dehydrogenase assay were performed on chenodeoxycholic acid or gastroesophageal reflux contents exposed to esophageal squamous cell carcinoma cell line. RESULTS: Sixty-seven percent of the controls (n = 12) developed esophageal cancer, but animals that received TJ-14 (n = 10) had a cancer incidence of 10% (P=.007). Barrett's metaplasia was found in 83% of the rats in the control group and 50% of the rats in the TJ-14 indicating a protective tendency of TJ-14 (P=.095). All of the rats developed proliferative hyperplasia. The number of M2 phenotype macrophage were significantly decreased in the TJ-14 group compared to the control group in both Barrett's metaplasia and esophageal cancer lesions. TJ-14 inhibited chenodeoxycholic acid or gastroesophageal reflux content-induced prostaglandin E2 production in esophageal squamous cell carcinoma cell. CONCLUSION: TJ-14 reduced the incidence of reflux-induced esophageal cancer and the infiltration of M2 macrophages in a surgical rat model or suppressed prostaglandin E2 production in esophageal squamous cell carcinoma cell. Further investigation is required regarding the potential clinical use of TJ-14 as an esophageal cancer chemopreventive agent.

Neoadjuvant Chemotherapy with Gemcitabine Plus Nab-paclitaxel Reduces the Number of Cancer-associated Fibroblasts Through Depletion of Pancreatic Stroma.

BACKGROUND: In this study, the effects of neoadjuvant chemotherapy (NAC) on cancer-associated fibroblasts (CAFs) in pancreatic cancer stroma were investigated. MATERIALS AND METHODS: Density of α-smooth muscle actin (αSMA)-positive fibroblasts in resected surgical specimens from untreated patients, patients receiving conventional gemcitabine plus S-1 (GS), and patients receiving gemcitabine plus nab-paclitaxel (GnP) was determined by hybrid cell counting. 18F-Fluorodeoxyglucose positron-emission tomography (FDG-PET) scans and carbohydrate antigen 19-9 (CA19-9) concentrations were used to assess tumor activity before and after chemotherapy in the GnP group. RESULTS: In this retrospective study of 65 patients, αSMA expression was reduced in the GnP group, as revealed by markedly disorganized collagen and a low density of αSMA-positive fibroblasts. There were significantly fewer αSMA-positive fibroblasts in the GnP than in the untreated and GS groups, but there was no significant difference between the latter two groups. αSMA density reflected a decrease in standardized uptake value on FDG-PET, but not CA19-9 concentration, after GnP chemotherapy. CONCLUSION: These data suggest that the GnP regimen induces stromal depletion, resulting in fewer CAFs.

Low-dose valproic acid with low-dose gemcitabine augments MHC class I-related chain A/B expression without inducing the release of soluble MHC class I-related chain A/B.

To improve natural killer group 2 member D (NKG2D)-dependent cytotoxicity, the inhibition of cleavage and release of major histocompatibility complex class 1-related chain (MIC) molecules from the tumor surface are required. Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, is able to induce cell-surface MICA/B on tumor cells. In the present study, the ability of VPA and gemcitabine (GEM) to upregulate MICA/B in pancreatic cancer cells was investigated, resulting in the inhibition of cleavage and release of MIC molecules from the tumor surface. Flow cytometry was used to quantify MICA/B expression in six human pancreatic cancer lines. Functional cytotoxic activity of γδT cells against pancreatic cancer cells treated with VPA and GEM was determined using cytotoxicity assays. At low doses of VPA (0.7 mM) and GEM (0.001 µM), which did not induce tumor growth alterations, the agents individually increased cell-surface MICA/B expression in MICA/B-positive cell lines, but not in the MICA/B-negative cell line. Furthermore, the combination of VPA and GEM synergistically induced cell-surface MICA/B expression. In MICA/B-positive cell lines, the increase in MICA/B expression was dependent on VPA concentration. The combination of low-dose VPA and GEM enhanced the susceptibility of the PANC-1 cell line to γδT cell-mediated tumor cell lysis. It was observed that soluble MIC was released from PANC-1 in the culture supernatant following treatment with GEM. However, the combination of low-dose VPA with low-dose GEM increased MICA/B expression without inducing soluble MIC, resulting in enhanced tumor cell lysis. The results of the present study suggest that the combined administration of low-dose VPA with low-dose GEM has the potential to enhance the therapeutic effects of immunotherapy in pancreatic cancer. Furthermore, it is proposed that the combination acts, in part, by upregulating MICA/B and prevents soluble MIC from being released.

Low-dose gemcitabine induces major histocompatibility complex class I-related chain A/B expression and enhances an antitumor innate immune response in pancreatic cancer.

We investigated the effect of gemcitabine (GEM), a key drug for pancreatic cancer treatment, on the expression of cell surface MICA/B in pancreatic cancer cells and resulting cytotoxicity of γδ T cells. We assessed the effect of GEM on the upregulation of cell surface MICA/B expression by flow cytometry, utilizing six pancreatic cancer cell lines. MICA and CD16 expressions from resected pancreatic cancer patient specimens, which received neoadjuvant chemotherapy (NAC) with GEM, were analyzed by immunohistochemistry. GEM could increase MICA/B expression on cell surface in pancreatic cancer cell lines (in 2 of 6 cell lines). This effect was most effectively at concentration not affecting cell growth of GEM (0.001 µM), because MICA/B negative population was appeared at concentration at cytostatic and cytotoxic effect to cell growth (0.1 and 10 µM). The cytotoxic activity of γδ T cells against PANC-1 was detected and functions through interactions between NKG2D and MICA/B. However, the enhancement of NKG2D-dependent cytotoxicity with increased MICA/B expression, by GEM treatment, was not observed. In addition, soluble MIC molecules were released from pancreatic cancer cell lines in culture supernatant with GEM treatment. Immunohistochemical staining demonstrated that MICA expression in tumor cells and CD16 positive cells surrounding tumors were significantly higher in the NAC group compared to that of the control group. There was a significant correlation between NAC and MICA expression, as well as NAC and CD16 positive cell expression. The present results indicate that low-dose GEM-induced MICA/B expression enhances innate immune function rather than cytotoxicity in pancreatic cancer. In addition, our result suggests that the inhibition of cleavage and release of MIC molecules from the tumor surface could potentially improve NKG2D-dependent cytotoxicity.

A Three-phase Approach for the Early Identification of Acute Lung Injury Induced by Severe Sepsis.

A number of studies have reported that acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are independent risk factors for organ dysfunction and mortality in patients with sepsis. Although ALI/ARDS might be an essential therapeutic target during the management of sepsis, severe sepsis should be treated effectively and as soon as identified. We have classified three phases, ranging from sepsis to organ dysfunction, characterizing the interaction between neutrophils and platelets. The first phase is neutrophil extracellular trap (NET) formation and intravasated platelet aggregation. The next phase is extravasated platelet aggregation (EPA), promoted by NET-facilitated detachment of endothelial cells. The final phase is organ dysfunction, caused by pulmonary veno-occlusive disease (VOD), fibrosis, and immunoparalysis induced by EPA. Severe sepsis is characterized by a continuum of coagulopathy, with coagulation abnormalities often developing before the onset of clinical symptoms. The initial medical treatment for ALI/ARDS is inhibition of NET formation and intravasated platelet aggregation to prevent endothelial cell damage (Phase 1). Beraprost and silvestat, phosphodiesterase 3 (PDE3) inhibitors, are often administered in clinical practice. To determine hypercoagulopathy, plasma levels of thrombin-antithrombin complex and plasmin-plasmin inhibitor complex are continuously monitored in patients with suspected sepsis. Furthermore, the implementation of quality indicators for the early management of severe sepsis and septic shock is strongly associated with a reduced mortality. We conclude that pathophysiology of organ dysfunction from severe sepsis is caused by pulmonary VOD, fibrosis, and EPA-facilitated immunoparalysis. In order to prevent ALI/ARDS in patients with sepsis, countermeasures for NET and platelet aggregation should be pre-emptively employed and confirmed by several trials.