Strategy of Patient-Specific Therapeutics in Cardiovascular Disease Through Single-Cell RNA Sequencing

Recently, single cell RNA sequencing (scRNA-seq) technology has enabled the discovery of novel or rare subtypes of cells and their characteristics. This technique has advanced unprecedented biomedical research by enabling the profiling and analysis of the transcriptomes of single cells at high resolution and throughput. Thus, scRNA-seq has contributed to recent advances in cardiovascular research by the generation of cell atlases of heart and blood vessels and the elucidation of mechanisms involved in cardiovascular development and diseases. This review summarizes the overall workflow of the scRNA-seq technique itself and key findings in the cardiovascular development and diseases based on the previous studies. In particular, we focused on how the single-cell sequencing technology can be utilized in clinical field and precision medicine to treat specific diseases.

The Key Role of DNA Methylation and Histone Acetylation in Epigenetics of Atherosclerosis

Atherosclerosis, which is the most common chronic disease of the coronary artery, constitutes a vascular pathology induced by inflammation and plaque accumulation within arterial vessel walls. Both DNA methylation and histone modifications are epigenetic changes relevant for atherosclerosis. Recent studies have shown that the DNA methylation and histone modification systems are closely interrelated and mechanically dependent on each other.Herein, we explore the functional linkage between these systems, with a particular emphasis on several recent findings suggesting that histone acetylation can help in targeting DNA methylation and that DNA methylation may control gene expression during atherosclerosis.

PPARgamma modulates vascular smooth muscle cell phenotype via a protein kinase G-dependent pathway and reduces neointimal hyperplasia after vascular injury

Vascular smooth muscle cells (VSMCs) undergo phenotypic changes in response to vascular injury such as angioplasty. Protein kinase G (PKG) has an important role in the process of VSMC phenotype switching. In this study, we examined whether rosiglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma agonist, could modulate VSMC phenotype through the PKG pathway to reduce neointimal hyperplasia after angioplasty. In vitro experiments showed that rosiglitazone inhibited the phenotype change of VSMCs from a contractile to a synthetic form. The platelet-derived growth factor (PDGF)-induced reduction of PKG level was reversed by rosiglitazone treatment, resulting in increased PKG activity. This increased activity of PKG resulted in phosphorylation of vasodilator-stimulated phosphoprotein at serine 239, leading to inhibited proliferation of VSMCs. Interestingly, rosiglitazone did not change the level of nitric oxide (NO) or cyclic guanosine monophosphate (cGMP), which are upstream of PKG, suggesting that rosiglitazone influences PKG itself. Chromatin immunoprecipitation assays for the PKG promoter showed that the activation of PKG by rosiglitazone was mediated by the increased binding of Sp1 on the promoter region of PKG. In vivo experiments showed that rosiglitazone significantly inhibited neointimal formation after balloon injury. Immunohistochemistry staining for calponin and thrombospondin showed that this effect of rosiglitazone was mediated by modulating VSMC phenotype. Our findings demonstrate that rosiglitazone is a potent modulator of VSMC phenotype, which is regulated by PKG. This activation of PKG by rosiglitazone results in reduced neointimal hyperplasia after angioplasty. These results provide important mechanistic insight into the cardiovascular-protective effect of PPARgamma.

Angiopoietin-1 Protects Endothelial Cells From Hypoxia-Induced Apoptosis via Inhibition of Phosphatase and Tensin Homologue Deleted From Chromosome Ten

BACKGROUND AND OBJECTIVES: Angiopoietin-1 (Ang1) is a regulator of blood vessel growth and maturation, and prevents radiation-induced or serum deprivation-induced apoptosis. Phosphatase and tensin homologue deleted from chromosome ten (PTEN), a well-known tumor suppressor, regulates cell cycle arrest and apoptosis. Hypoxia induces apoptosis by increasing the expression of PTEN. We hypothesized that Ang1 may regulate PTEN expression and, thus, reduce endothelial apoptosis under hypoxia in vitro and in vivo. Materials and METHODS: In vitro, human umbilical vein endothelial cells (HUVECs) were treated with Ang1, and signaling pathways were investigated. In vivo, eight-week-old C57BL/6 mice were used for a hind limb ischemia model. Ang1 or normal saline was intramusculary injected. Blood flow was evaluated by a laser Doppler perfusion analyzer and tissue histology. RESULTS: The expression of PTEN was markedly upregulated in HUVECs after hypoxic stimulation, whereas Ang1 suppressed PTEN expression. Tie2-Fc, a soluble form of Tie2 (sTie2) that blocks Ang1, reversed the Ang1 effect on PTEN reduction under hypoxia. Ang1 inhibited the nuclear translocation of nuclear transcription factor-kB (NF-kB), a binding factor for the PTEN promoter and Foxo1. Hypoxia-induced p27 expression and apoptosis were also suppressed by Ang1. In the mouse hind limb ischemia model, we observed a high capillary density, numerous proliferating cells and diminished cell death in skeletal muscle tissue in the Ang1 injected group. CONCLUSION: Ang1 enhanced endothelial cell survival by reducing apoptosis via PTEN down-regulation in HUVECs under hypoxia. Local injection of Ang1 significantly reduced apoptotic cells in vivo, and prevented limb loss for ischemic hind limb mice. Thus, Ang1 may be an effective therapeutic for protection from ischemic-endothelial cell injury.

Generalized Pustular Psoriasis and Hepatic Dysfunction Associated with Oral Terbinafine Therapy

We report a case of 61-yr-old man with stable psoriasis who progressively developed generalized pustular eruption, erythroderma, fever, and hepatic dysfunction following oral terbinafine. Skin biopsy was compatible with pustular psoriasis. After discontinuation of terbinafine and initiating topical corticosteroid and calcipotriol combination with narrow band ultraviolet B therapy, patient's condition slowly improved until complete remission was reached 2 weeks later. The diagnosis of generalized pustular psoriasis (GPP) induced by oral terbinafine was made. To our knowledge, this is the first report of GPP accompanied by hepatic dysfunction associated with oral terbinafine therapy.

SADBE (Squaric acid dibutylester) Immunotherapy in Alopecia Areata / 대한피부과학회지

BACKGROUND: To date, highly variable results for use of topical Squaric acid dibutylester (SADBE) in the treatment of alopecia areata have been reported. Furthermore, there are no reports on SADBE in Korean dermatologic literature yet. OBJECTIVE: The purpose of this study was to evaluate the efficacy and the tolerability of SADBE in the treatment of severe alopecia areata. METHOD: A total of 22 cases of severe alopecia areata were enrolled in this study. After sensitization of the patients with 2% SADBE in acetone, the subsequent on-going treatments were done with 0.00001% to 2% SADBE with an interval of 1 to 2 weeks. The sensitization rate, the therapeutic efficacy and side effects of SADBE during the treatment course were evaluated. The efficacy was evaluated by 5 rating scales and we continued to check the recurrence of the lesions in the patients who had shown complete regrowth. RESULTS: The mean sensitization rate was 1.55. The treatment frequency at the time of initial hair regrowth ranged from 5 to 21 (mean-10.2). In the 22 patients who were treated for 6 months, more than 90% regrowth in 10 patients (45.5%) was observed, good or fair results (50-89% regrowth) in 3 patients (13.6%), and less than 49% regrowth in 9 patients (40.9%). In this study, only the duration of disease and being recurrent or not, among many prognostic factors, were statistically significant (p<0.05, chi2 -test). In half of the patients, various side effects were observed. Most common side was severe eczema at the sensitization site. Side effects during the treatment course were as follows; severe contact dermatitis, remote dermatitis, generalized pruritus, lymphadenopathy, and dermographism. But the result of side effects was not enough to give up treatment. Of the 10 patients who showed more than 90% regrowth, 4 patients had a recurrence of the lesion in their follow-up period (mean-4.75 months). CONCLUSION: The SADBE immunotherapy is effective and well-tolerated in Korean patients with severe alopecia areata.

The Effects of 5% Imiquimod Cream on Verruca Plana / 대한피부과학회지

BACKGROUND: Verruca plana is a cutaneous human papillomavirus (HPV) infections. Although various treatments such as destructive methods or immunomodulating agents have been used, none are uniformly effective or prevent recurrence. Ideal treatment for verruca plana should target on an increasing local immune response to the HPV infection. Recently, imiquimod, a topical immune- response modifier, has been successfully used in the treatment of external anogenital warts. OBJECTIVE: The aim of this study was to determine the efficacy and safety of 5% imiquimod cream for the treatment of verruca plana. METHOD: Seven patients with verruca plana were treated with 5% imiquimod cream 3 times a week. at night, for 16 weeks or until complete clearance of lesions had occured. During the follow- up period, the onset time of effects, clearance rate, side effects, and recurrence rate were recorded. At 16 weeks after treatment, a clearance rate was determined by a 3 scale rating; complete-100% clearance / partial-less than 100% clearance / failure-no clearance. RESULTS: The onset time of effects ranged from 1 to 4 weeks (mean-1.7 weeks). The clearance rate at 16 weeks after treatment were as follows; complete-4 (57.1%), partial-2 (28.6%), and failure - 1 (14.2%). No patient showed systemic side effects or long-term adverse effects such as pigmentary disorders or scarring. In the subjective local skin reactions, itching was the only symptom and was common (4/7, 57.1%). With objective skin reactions, erythema was the most common (4/7, 57.1%), followed by erosion and scabbing (2/7, 28.6%). In long-term follow-up of those patients who showed complete clearance, no one encountered recurrence. CONCLUSION: This data demonstrates that 5% imiquimod cream is an effective and promising treatment modality for verruca plana. Because it is non-destructive, safe, and easy to use, it resulted in an excellent cosmetic outcome.

Reactivity to Autologous Serum Skin Test and Clinical Features in Chronic Idiopathic Urticaria / 대한피부과학회지

BACKGROUND: Although the pathogenesis of chronic idiopathic urticaria (CIU) has not yet been fully elucidated, previous studies have identified functional, histamine-releasing autoantibodies against either alpha subunit of the high affinity IgE receptor (Fc epsilon RI alpha) or IgE in the serum of some patients with CIU. Therefore an autologous serum skin test (ASST) can be used as a predictive clinical test to determine the presence of circulating histamine-releasing factors in the serum of CIU patients, and there are some reports stating that patients with positive ASST tend to have more severe symptoms of urticaria than patients with a negative result. OBJECTIVE: This study was designed to determine the incidence of positive ASST in CIU patients and examine whether there are significant differences in the clinical features and laboratory findings between groups of positive and negative responses to ASST. METHOD: We prospectively performed ASST and laboratory tests on 70 patients with CIU, and also checked clinical features. RESULTS: Intradermal injection of autologous serum or plasma induced a wheal and flare response in 44 out of 70 CIU patients (62.9%). However, no significant difference in the clinical features and laboratory findings, other than angioedema, was noted between ASST-positive and negative groups. CONCLUSION: According to our results, the incidence of positive ASST was high in CIU patients, but ASST is not helpful to predict the severity or clinical course of CIU.

Clinical Study of Drug Rash with Eosinophilia and Systemic Symptoms (DRESS) on Drug Eruption Patients Over the last 10 years (1995-2004) / 대한피부과학회지

BACKGROUND: Drug rash with eosinophilia and systemic symptoms (DRESS), previously named drug `hypersensitivity syndrome', is a subset of severe drug eruption with quite distinct clinical presentations. Perhaps because of its relatively late onset and variable presentations, the diagnosis of DRESS may be delayed. OBJECTIVE: This study was designed to determine the incidence and investigate the causative drugs and clinical characteristics of DRESS. METHOD: We retrospectively reviewed the clinical features and laboratory findings of DRESS in 795 drug eruption patients who had visited Pusan National University Hospital over the last 10 years (1995-2004). RESULTS: 1. Of 795 drug eruption patients, 14 (1.76%) received a diagnosis of DRESS. 2. The average age of onset was 44.5 years and there was no significant difference according to sex. 3. The most common causative agent of DRESS was carbamazepine (50%), followed by allopurinol, captopril, phenytoin and antituberculous medications. 4. DRESS developed 2-10 weeks after administration of the causative agent, and the average latent period was 4.6 weeks.

A Case of Tufted Angioma Arising within a Nevus Flammeus / 대한피부과학회지

Tufted angioma is an uncommon, slowly-progressive vascular tumor, found typically in infants, young children and sometimes at birth or during adulthood. It shows a characteristic histopathologic finding, the so-called "cannonball" appearance. Various tumors can be developed in the nevus flammeus, such as pyogenic granuloma, basal cell carcinoma, squamous cell carcinoma, giant proliferative hemangioma and lymphangioma circumscriptum. Tufted angioma can be also accompanied with nevus flammeus and the coexistence of tufted angioma and nevus flammeus is a very rare condition. We report a case of tufted angioma arising within nevus flammeus in the left axilla of a 47 year-old female.

A Study on Utilization of Alternative Medicine for Patients with Atopic Dermatitis / 대한피부과학회지

BACKGROUND: Alternative medicine - sometimes called complementary or supplementary medicine - may be defined as forms of therapy or examination that have no scientific basis and whose effect or diagnostic reliability has not been demonstrated by scientific methods. Recently, alternative medicine has been used in various chronic diseases including atopic dermatitis (AD) and has attracted attention in the mass media. Several studies on the use of alternative medicine in patients with AD have been performed in western countries, however only a few studies have been conducted in Korea. OBJECTIVE: The purpose of this study was to investigate the use of alternative medicine in AD patients. METHOD: A total of 100 patients with AD were enrolled on the study, and interviewed with a questionnaire about their past history of AD and the use of alternative medicine. RESULTS: The results obtained are summarized as follows: 1. 84 out of 100 patients (84.0%) reported previous or current use of more than one type of alternative medicine. 2. The most common type of alternative medicine used was herbal remedies (73.8%). Spa and bath therapies (47.6%), health food preparations (39.3%) and diet therapy (25.0%) were also commonly used. 3. The frequency of alternative medicine used was related to onset and severity of AD. 4. The most common reason for using alternative medicine was `I wish to try everything' (60.2%), and the most common source of information on alternative medicine was relatives and friends who did not have the disease (41.4%). 5. The therapeutic effect of alternative medicine was found to be excellent in 25.3% of patients, but no change was seen in 58.6% of patients. 6. The most common side effect of alternative medicine was aggravation of symptoms. Other side effects included urticaria, diarrhea, fever and chills. 7. The average monthly cost for alternative medicine was 210, 000 won/person. CONCLUSION: The use of various types of alternative medicine in patients with AD is very common. However, these tend to be used impulsively and without caution or adequate knowledge. Therefore, dermatologists need to be aware of the benefits and adverse effects of alternative medicines.

The Expression of VEGF in HaCaT Cell Induced by Pressure and Its Role in the Pathogenesis of Psoriasis / 대한피부과학회지

BACKGROUND: Psoriasis is a common, chronic skin disease characterized by recurrent erythematous skin plaque. Psoriatic lesion of patients exibits hyperproliferation of epidermis, a variable inflammatory cell infiltrate, and abnormalities of the papillary dermal blood vessels. Expansion of the dermal microvasculature and increased expression of VEGF in keratinocytes are prominent features of psoriasis. The normal appearing skin of psoriatic patients may respond to an injury with the appearance of a psoriatic lesion. It has been demonstrated that trauma or pressure on the skin develop psoriasis from normal appearing skin of psoriatic patients and temporary obligation of the vessels prevented the `Koebner phenomenon'. These clinical observations indicate the importance of the vascular compartment in the initiation of the psoriatic lesion. OBJECTIVE: We investigated the relationship between expression of VEGF and pressure in the development of Koebner phenomenon. METHOD: Metal sinker (10g) was added to the cultured HaCaT cells for 24 hours and the expression of VEGF was checked by immunohistochemistry. RESULTS: HaCaT cells pressured by metal sinker showed increased expression of VEGF compared to control cells. CONCLUSION: These results suggest that pressure probably induces the Koebner phenomenon through VEGF-induced angiogenesis.

Insulin-like Growth Factor-II (IGF-II) Induces Vascular Endothelial Growth Factor (VEGF) Expression through IGFR-1 and ERK1/2 Activation in Psoriasis / 대한피부과학회지

BACKGROUND: Psoriasis is a chronic relapsing and angiogenic skin disease characterized by variable clinical features. But the pathogenetic process resulting in vascular morphological changes remains to be proven. It is reported that the potent angiogenic factor VEGF is overexpressed in psoriatic epidermis and the level of IGF-II is significantly elevated in tissue fluid and serum of the psoriatic lesion. OBJECTIVE AND METHOD: To find the mechanism of VEGF induction in pathogenesis of psoriasis, we adopt IGF-II as a paracrine inducer of VEGF in psoriasis. To investigate the signaling pathway of IGF-II-induced VEGF, we determined ERK1/2 activity in IGF-II-treated psoriatic cells. RESULT: In this report, we demonstrated that IGF-II induced the expression of VEGF in lesional keratinocytes of psoriasis. And IGF-II stimulated the expression of its receptor, IGFR-I in psoriatic cells. Treatment of anti-IGFR-I neutralizing antibody diminished VEGF mRNA level induced by IGF-II, indicating that VEGF induction by IGF-II may be mediated through IGFR-I. By the treatment of PD98059, specific inhibitor of upstream ERK activator MAP kinase/ERK kinase (MEK), the expression of VEGF induced by IGF-II was dramatically reduced. CONCLUSION: Taken together, these results suggest that IGF-II might regulate angiogenesis by the induction of VEGF through the MAP kinase pathway mediated by IGFR-I in the lesion of psoriasis.

Studies on the Mechanism of Hypoxic Increase of VEGF Expression in the Hep3B Human Hepatoma Cells / 대한암학회지

PURPOSE: Hepatocellular carcinoma (HCC), a typical hypervasculized tumor is very sensitive to hypoxia and vascular endothelial growth factor (VEGF) has previously been identified to be up-regulated in response to hypoxia in several cell types. However, the molecular mechanisms by which hypoxia is sensed by the cells remain enigmatic. To investigate whether calcium and AP-1 are involved in hypoxia-sensing mechanism, we performed following experiments. MATERIALS AND METHODS: Hep3B cells were grown in hypoxic condition. To assess cell viability, MTT assay was performed. To investigate the effect of calcium and AP-1, northern blot analysis was performed after treatment with BAPTA/AM. RESULTS: The expression of VEGF was significantly up-regulated by hypoxia in Hep3B, hepatocellular carcinoma cell line. The increased expression of VEGF induced by hypoxia was blocked by the addition of BAPTA/AM, a cytosolic calcium chelator to the media. In addition, we found that the expression of c-jun protooncogene was also up-regulated by hypoxia. Hypoxic increase of c-jun expression was also normalized by the treatment with BAPTA/AM. CONCLUSION: These results suggest that the increased expression of VEGF by hypoxia is mediated through the calcium and c-jun signalling pathway in the Hep3B human hepatoma cell lines.