Validation of Human Bone Marrow-derived Mesenchymal Stem Cells and MCF-7 Breast Cancer Cells Co-culture Using a 3D Perfused Biomimetic Microfluidic Platform.

BACKGROUND/AIM: Microfluidic experimental models allow to study the mutual interrelation between tumor development and the microvasculature avoiding animal use and lacking interspecies differences. This study aimed to develop and characterize a 3D tissue culture model employing a two-compartment microfluidic chip-perfused platform to visualize and quantify human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and MCF-7 breast cancer cell-cell interactions in real time. MATERIALS AND METHODS: MCF-7 cells were implanted in the tumor chamber and hBM-MSCs were injected into microvascular channels. hBM-MSCs culture media was perfused into microvascular compartments. The microfluidic device was microscopically examined weekly for four weeks. RESULTS: VE- and E-cadherin immunofluorescence validated hBM-MSCs differentiation into endothelial cells and MCF-7 cell tumor formation. hBM-MSCs differentiation was highly heterogeneous along the microvascular channels, due to different perfusion flow. hBM-MSCs lining microvascular channels acquired VE-cadherin positive endothelial phenotype and continuously covered microchannels as an endothelium like layer. MCF-7 cells were constantly grown as spheroidal aggregates and later formed a compact area of E-cadherin-positive tumor cells inside tumor compartment. CONCLUSION: Our study provides valuable knowledge on the properties of hBM-MSCs as vasculogenesis-supporting cells when co-cultured with MCF-7 cells on a 3D perfused biomimetic microfluidic device. This newly established model may serve as an experimental platform for testing anti-tumor/anti-angiogenic drugs.

Artificial Intelligence (AI) Based Analysis of In Vivo Polymers and Collagen Scaffolds Inducing Vascularization.

BACKGROUND/AIM: Biomaterials are essential in modern medicine, both for patients and research. Their ability to acquire and maintain functional vascularization is currently debated. The aim of this study was to evaluate the vascularization induced by two collagen-based scaffolds (with 2D and 3D structures) and one non-collagen scaffold implanted on the chick embryo chorioallantoic membrane (CAM). MATERIALS AND METHODS: Classical stereomicroscopic image vascular assessment was enhanced with the IKOSA software by using two applications: the CAM assay and the Network Formation Assay, evaluating the vessel branching potential, vascular area, as well as tube length and thickness. RESULTS: Both collagen-based scaffolds induced non-inflammatory angiogenesis, but the non-collagen scaffold induced a massive inflammation followed by inflammatory-related angiogenesis. Vessels branching points/Region of Interest (Px^2) and Vessel branching points/Vessel total area (Px^2), increased exponentially until day 5 of the experiment certifying a sustained and continuous angiogenic process induced by 3D collagen scaffolds. CONCLUSION: Collagen-based scaffolds may be more suitable for neovascularization compared to non-collagen scaffolds. The present study demonstrates the potential of the CAM model in combination with AI-based software for the evaluation of vascularization in biomaterials. This approach could help to reduce and replace animal experimentation in the pre-screening of biomaterials.

Age, Sex, Metabolic and Pharmacologic Factors May Predict Nonresponse Status to Rheumatoid Arthritis Therapies.

BACKGROUND/AIM: A real challenge for patients with rheumatoid arthritis (RA) and rheumatologists is primary nonresponse status (PNRS) or secondary nonresponse status (SNRS) to various therapies. Despite their detrimental influence on patient life quality, PNRS and SNRS have no accurate definition and no early predictive criteria for their development exist. Patients with RA under 40 years of age are rare, hence PNRS and SNRS data for this age group are scarce. This study examined the PNRS and SNRS according to sex, age, BMI, therapy type, and duration. PATIENTS AND METHODS: Retrospectively, 115 patients with RA having PNRS and/or SNRS were stratified by age (22-39, 40-59, and 60-81). The association between body mass index (BMI), proinflammatory cytokines inhibitors, JAK inhibitors, and TNF-alpha inhibitors, sex, age, and PNRS and SNRS was examined. RESULTS: All three proinflammatory cytokine inhibitors (rituximab, tocilizumab, and abatacept) were associated with PNRS and SNRS in women with a high BMI aged 40-59 years. Abatacept-related PNRS and SNRS was significant in women with normal BMI aged 60-81 years. Adalimumab, infliximab, and golimumab affected SNRS differently in women with normal BMI aged 22-39 years and women with high BMI aged 60-81 years. Etanercept and infliximab were associated with SNRS status in men with high-BMI aged 40-59 years. CONCLUSION: PNRS and SNRS development in patients with RA is significantly influenced by age, sex, and BMI, but most importantly is closely and differentially related to therapy type and duration.

Reassessing Breast Cancer-Associated Fibroblasts (CAFs) Interactions with Other Stromal Components and Clinico-Pathologic Parameters by Using Immunohistochemistry and Digital Image Analysis (DIA).

BACKGROUND: Breast cancer (BC) stroma has CD34- and αSMA-positive cancer-associated fibroblasts (CAFs) differently distributed. During malignant transformation, CD34-positive fibroblasts decrease while αSMA-positive CAFs increase. The prevalence of αSMA-positive CAFs in BC stroma makes microscopic examination difficult without digital image analysis processing (DIA). DIA was used to compare CD34- and αSMA-positive CAFs among breast cancer molecular subgroups. DIA-derived data were linked to age, survival, tumor stroma vessels, tertiary lymphoid structures (TLS), invasion, and recurrence. METHODS: Double immunostaining for CD34 and αSMA showed different CAF distribution patterns in normal and BC tissues. Single CD34 immunohistochemistry on supplemental slides quantified tumor stroma CD34_CAFs. Digital image analysis (DIA) data on CAF density, intensity, stromal score, and H-score were correlated with clinico-pathologic factors. RESULTS: CD34/αSMA CAF proportion was significantly related to age in Luminal A (LA), Luminal B (LB), and HER2 subtypes. CD34_CAF influence on survival, invasion, and recurrence of LA, LB-HER2, and TNBC subtypes was found to be significant. The CD34/αSMA-expressing CAFs exhibited a heterogeneous impact on stromal vasculature and TLS. CONCLUSION: BC stromal CD34_CAFs/αSMA_CAFs have an impact on survival, invasion, and recurrence differently between BC molecular subtypes. The tumor stroma DIA assessment may have predictive potential to prognosis and long-term follow-up of patients with breast cancer.

Tertiary Lymphoid Structures (TLSs) and Stromal Blood Vessels Have Significant and Heterogeneous Impact on Recurrence, Lymphovascular and Perineural Invasion amongst Breast Cancer Molecular Subtypes.

BACKGROUND: Tertiary lymphoid structures (TLSs) mediate local antitumor immunity, and interest in them significantly increased since cancer immunotherapy was implemented. We examined TLS- tumor stromal blood vessel interplay for each breast cancer (BC) molecular subtype related to recurrence, lymphovascular invasion (LVI), and perineural invasion (PnI). METHODS: TLSs were quantified on hematoxylin and eosin stain specimens followed by CD34/smooth muscle actin (SMA) double immunostaining for stromal blood vessel maturation assessment. Statistical analysis linked microscopy to recurrence, LVI, and PnI. RESULTS: TLS negative (TLS-) subgroups in each BC molecular subtype (except to Luminal A) have higher LVI, PnI, and recurrence. A significant rise in LVI and PnI were observed for the HER2+/TLS- subgroup (p < 0.001). The triple negative breast cancer (TNBC)/TLS- subgroup had the highest recurrence and invasion risk which was also significantly related to tumor grade. PnI but not LVI significantly influenced recurrence in the TNBC/TLS+ subgroup (p < 0.001). TLS-stromal blood vessel interrelation was different amongst BC molecular subtypes. CONCLUSION: BC invasion and recurrence are strongly influenced by TLS presence and stromal blood vessels, especially for HER2 and TNBC BC molecular subtypes.

"Face(s)" of a PHACE(S) Syndrome Patient before and after Therapy: Particular Case Report and Review of Literature.

A rare, uncommon disorder called PHACE(S) (P-posterior fossa anomalies, H-hemangioma, A-arterial anomalies, C-cardiac anomalies, E-eye anomalies, and S-sternal cleft) of unknown etiology was rarely reported. Children are susceptible to developing PHACE(S) syndrome from the moment they are born. It may be challenging for a physician to appropriately diagnose and treat children with PHACE due to the multifaceted nature of the disease and the extensive range of consequences that may be associated with it. A one-month-old newborn girl was admitted to hospital with extensive, multiple facial infantile hemangiomas, ulceration of the lower lip hemangioma-like lesion, cardiovascular, sternal, and neurological concomitant malformations. Five days following the initial application of the medication, systemic treatment with propranolol and topical treatment with silver sulfadiazine produced their first noticeable benefits. The lip ulceration was mostly healed and facial hemangioma started to regress. The regression continued under therapy and this effect persists for 6 months since Propranolol therapy ended. No cardiovascular or neurological clinical events have been registered during follow-up. The present case has three peculiarities: (1) high number of facial hemangiomas; (2) presence of subependymal cyst not yet reported in the literature associated with PHACE syndrome; and (3) lack of cardiovascular events during therapy knowing that these events frequently appear in PHACE syndrome patients.

The Mutually Mediated Chloride Intracellular Channel Protein 1 (CLIC1) Relationship between Malignant Cells and Tumor Blood Vessel Endothelium Exhibits a Significant Impact on Tumor Angiogenesis, Progression, and Metastasis in Clear Cell Renal Cell Carcinoma (ccRCC).

Background: Overexpression of chloride intracellular channel protein 1 (CLIC1) in tumor cells has been confirmed, but it has received less attention in the tumor blood vessel endothelium. Aim: The assessment of CLIC1 expression in ccRCC tumor blood vessels and its relationship with TNM parameters and tumor cell CLIC1 expression. Methods: CLIC1 immunostaining in ccRCC was evaluated in 50 cases in both malignant cells and tumor blood vessels (CLIC1 microvessel density-CLIC1-MVD) and was correlated with TNM staging parameters. Results: CLIC1-MVD was observed in approximately 65% of cases, and CLIC1 co-localization in both tumor and endothelial cells was observed in 59% of cases. ccRCC was classified into four groups (Classes 0−3) based on the percentage of positive tumor cells, with each group including sub-groups defined by CLIC1 expression in the endothelium. Class 3 (60−100% positive tumor cells) had the highest CLIC1-MVD, with an impact on T and M parameters (p value = 0.007 for T, and p value = 0.006 for M). For cases with CLIC1 intracellular translocation, there was a strong correlation between CLIC1-MVD and M (p value < 0.001). Conclusions: Co-expression of ccRCC tumor and endothelial cells promotes tumor progression and metastasis and should be investigated further as a potential therapeutic target for ccRCC and other human malignancies.

VEGF Pathway Gene Expression Profile of Proliferating versus Involuting Infantile Hemangiomas: Preliminary Evidence and Review of the Literature.

Background. Infantile hemangiomas may have unexpected behavior. Initial regression (spontaneously or drug-induced) may be followed by unexplained recurrences. At this moment, there are no well-established criteria to predict infantile hemangioma reccurrences. Methods. We compared the VEGF pathway gene expression profile for one case of involuting infantile hemangioma versus one case of recurrent proliferative infantile hemangioma using TaqMan Array. Results. We found ten genes upregulated for both involuting and recurrent proliferative hemangiomas: ACTB, KRAS, MAP2K1, HRAS, NOS3, BAD, HSPB1, HPRT1, GUSB, and CASP9. Thirteen genes were downregulated for both involuting and proliferative hemangiomas: FIGF, ACTG1, GRB2, MAPKAPK2, ACTG2, MAP2K2, MAPK3, HSP90AA1, MAP2K6, NRAS, ACTA1, KDR, and MAPK1. Three genes showed divergent expression between proliferating and involuting hemangiomas. Proliferating hemangioma had MAPK14 and AKT1 gene upregulation and ACTA2 downregulation. Involuting infantile hemangioma was characterized by ACTA2 upregulation and AKT1 and MAPK14 downregulation. Conclusions. Three genes, AKT1, p38/MAPK14, and ACTA2, were found to have divergent expression in proliferating and involuting infantile hemangiomas. Excepting AKT1, which was mentioned in the last ISSVA classification (strictly related to Proteus Syndrome), none of the other genes were reported. An accurate gene expression profile mapping of infantile hemangiomas together with a gene expression-based hemangioma classification is stringently needed.

Heterogeneity of Platelet Derived Growth Factor Pathway Gene Expression Profile Defines Three Distinct Subgroups of Renal Cell Carcinomas.

BACKGROUND/AIM: We previously described four different vascular patterns (reticular, diffuse, fasciculate, and trabecular) in renal cell carcinoma (RCC) suggesting an early and heterogeneous acquisition of perivascular cells most probably due to a particular PDGF pathway gene expression profile. The aim of the study was to study PDGF pathway gene expression profiles, separately for each vascular pattern. MATERIALS AND METHODS: TaqMan assay for the PDGF pathway was performed on twelve cases of ccRCC previously evaluated by histopathology, immunohistochemistry, and RNAscope. Gene expression profile was correlated with grade, invasion, vascular patterns, and VEGF. RESULTS: PIK3C3 and SLC9A3 genes were overexpressed in all vascular patterns, but they were significantly correlated with high VEGF mRNA in the reticular and diffuse pattern. STAT1, JAK2, SHC2, SRF and CHUK (IKK) were exclusively overexpressed in cases with diffuse vascular pattern. SLC9A3, CHUK and STAT3 were overexpressed in G2 tumors. CONCLUSION: Three ccRCC subgroups were defined: 1) PIK3C3 (VSP34)/SLC9A3 which may be proper for anti PIK3C3 inhibitors; 2) VEGFhigh subgroup where association of anti VEGF may be a benefit and 3) JAK2/STAT1 subgroup, potentially being eligible for anti JAK/STAT therapy associated with IKK inhibitors.

Anti-chloride Intracellular Channel Protein 1 (CLIC1) Antibodies Induce Tumour Necrosis and Angiogenesis Inhibition on In Vivo Experimental Models of Human Renal Cancer.

BACKGROUND/AIM: Chloride intracellular channel protein 1 (CLIC1) is known as a promoter of cancer progression, metastasis, and angiogenesis. Thus, CLIC1 could be a future therapeutic target. This study aimed to evaluate the effect of anti-CLIC1 antibodies on tumour cells and vessels of human renal cell carcinoma (RCC) in rabbit cornea and chick embryo chorioallantoic membrane (CAM) models. MATERIALS AND METHODS: Human cc-RCC xenografts on rabbit cornea and CAM surface were performed. Anti-CLIC1 antibodies were applied for 5 consecutive days on both tumor models. We comparatively evaluated treated and untreated tumors by combining ultrasonography with microscopic techniques. RESULTS: RCC implants rapidly recruited blood vessels and had an exponential growth rate on both tumor models. Anti-CLIC1 antibodies suppressed tumor growth by inducing tumor cell necrosis. Tumor vessels regressed rapidly but not completely during anti-CLIC1 antibodies based therapy. CONCLUSION: Anti-CLIC1 antibodies induced tumor necrosis and tumor vasculature regression in human cc-RCC xenografts in both in vivo experimental models.

Patterns of Tumor Vasculogenesis on a Chick Embryo Chorioallantoic Membrane In Vivo Model of Breast Cancer.

BACKGROUND/AIM: Understanding tumor vasculogenesis is a cornerstone for the inhibition of tumor progression. This study aimed to generate an in vivo breast cancer environment to analyze the patterns of tumor vasculogenesis. MATERIALS AND METHODS: Human mesenchymal stem cells (hMSC) and breast cancer MCF-7 cells (MCF-7) were seeded onto a chorioallantoic membrane (CAM) and, after a 7-day incubation, we performed a morphological and immunohistochemical analysis of CAM. RESULTS: hMSC and MCF-7 activated vasculogenesis and hematopoiesis on CAM. They stimulated the development of cord/capillary-like structures (CLS), formed by endothelial-like cells and hematopoietic cells. CLS presented a polygonal pattern, evolving towards a clearly visible plexus. Immunohistochemically, CLS were CD105+/AC133+/Oct3/4+, and the intensity was weak-moderate in the endothelial-like cells (inconstant) and weak in the hematopoietic cells. CONCLUSION: Tumor and embryonic vasculogenesis share a common paradigm, while CD105, AC133, and Oct3/4 were found to play a role in establishing the vasculogenic and hematopoietic stage.

Expression of ß1 adrenergic receptor in vascular anomalies in children.

OBJECTIVE: Controversial, heterogeneous, and inconsistent responses to beta-blockers have been reported in some cases of infantile proliferative hemangiomas. On the basis of these clinical observations, we aimed to examine the ß1 adrenergic receptor (ß1-AR) protein expression distribution among different types of pediatric vascular anomalies. METHODS: Immunohistochemistry (IHC) was performed for ß1-AR on 43 surgical specimens. RESULTS: We found positive ß1-AR IHC staining in all intramuscular hemangiomas, capillary-lymphatic, lymphatic, venous, and combined malformations, and Masson's tumor cases, as well as in 7 of 10 cases of proliferative infantile hemangiomas. CONCLUSIONS: Our research demonstrates, for the first time, the degree of heterogeneous expression of ß1-AR among pediatric vascular malformations. Our results support the need for ß1-AR assessment in pediatric vascular anomalies to select cases with a robust response to ß1-selective blockers. ß1-AR assessment may have a strong impact on therapeutic refinement for pediatric vascular anomalies by selecting cases with a stronger response to beta-blockers.

CLIC1 Expression in Skin Biopsies from Patients With Rheumatoid and Psoriatic Arthritis as a Potential Tool to Predict Therapy Response.

BACKGROUND/AIM: Chloride intracellular channel protein 1 (CLIC1) activates inflammasomes in rheumatoid (RA) and psoriatic (PsA) arthritis. We studied CLIC1 expression in RA and PsA patients' skin with vasculitis and its variability depending on the therapy used. MATERIALS AND METHODS: CLIC1 immunoexpression was evaluated in the vascular (CLIC1-V) and stromal (CLIC1-S) compartments of the RA and PsA skin biopsies of patients treated with methotrexate (MTX), leflunomid (LFN), corticotherapy (CT), or biological therapies. RESULTS: MTX significantly reduced CLIC1-S expression (p=0.016), whereas LFN decreased CLIC1-V (p<0.001). LFN therapy duration also correlated with CLIC1-V (p<0.001). CT decreased CLIC1-S expression (p=0.006). CLIC1-S expression persisted in skin biopsies despite of erythrocyte sedimentation rate (ESR, p=0.018) and C reactive protein (CRP, p=0.0026) normalisation. For PsA, CLIC1-S expression significantly related to MTX (p<0.022). Both CLIC1-S (p<0.001) and CLIC1-V (p=0.007) decreased by biological therapies in RA. CONCLUSION: CLIC1 expression is strongly influenced by the therapy used. Our data strongly support the extensive evaluation of CLIC1 in RA as a potential marker of inflammation and tool to predict therapy response.

Liver Metastatic Colorectal Tumor Cells Change Their Phenotype During Consecutive Passages on Chick Embryo Chorioallantoic Membrane: Lessons from the Lab to the Clinic.

BACKGROUND/AIM: Colon cancer liver metastases with desmoplastic growth pattern (dGP) have a highly heterogeneous therapy response. The aim of the study was to evaluate the dGP liver metastasis molecular profile from a chemo-naive patient by mimicking metastatic process on an experimental chick embryo chorioallantoic membrane (CAM) model. MATERIALS AND METHODS: Three successive CAM passages of dGP human colorectal liver metastases were immunophenotyped for keratin (K) 8, and 20, CLIC1, VEGF, EGFR, CD34, podoplanin, Ki67, E-cadherin and vimentin. RESULTS: Metastatic cells gradually lost K20 while K8, E-cadherin and vimentin heterogeneously increased during passages. VEGF, CLIC 1, EGFR expression increased in metastatic cells especially at the tumor graft periphery. Scattered proliferating and non-proliferating podoplanin-positive tumor cells, lymphatic and blood vessels were heterogeneously detected in tumor xenografts depending on passage stage. CONCLUSION: By mimicking repetitive metastatic processes we proved that metastatic cells change their phenotype. This may explain why not all metastases have a similar response to therapy.

Clinicopathological Features of Growth Hormone-producing Pituitary Adenomas and Correlation With Preoperative Laboratory Findings.

BACKGROUND/AIM: The histopathological variability of each type of pituitary adenoma (PA) that causes growth hormone (GH) excess influences the phenotype, radiological characteristics and therapy response of acromegaly patients. We correlated the immunohistochemical (IHC) features of GH-secreting PAs with their clinical, laboratory and imaging data. PATIENTS AND METHODS: We included 32 patients with documented acromegaly; tumour specimens were histologically and IHC examined: anterior pituitary hormones, pituitary-specific transcription factor-1 (PIT-1), Ki-67 labelling index were evaluated. RESULTS: Macroadenomas represented 93.75%. Post-surgery disease control negatively correlated with the maximum initial tumour diameter (p=0.04). Ki-67 did not predict remission. No correlation was found between GH serum levels and IHC expression (p=0.45). PIT-1 was positive in all specimens, two had a weak expression. Four were considered PIT-1 positive plurihormonal adenomas and several had unusual IHC combinations. CONCLUSION: PIT-1 accurately classifies GH-secreting PAs. The IHC classification as well as radiological dimensions and extent influence disease control, probably being the best prognosis factors.

Gene Expression Profile of Vascular Endothelial Growth Factors (VEGFs) and Platelet-derived Growth Factors (PDGFs) in the Normal Cornea.

BACKGROUND/AIM: Angiogenic growth factors expression is not known in the normal cornea. The aim was to study corneal gene expression profile of VEGF and PDGF pathways influencing the avascular state of cornea. MATERIALS AND METHODS: cDNA synthesis was performed from mRNA extracted from five fresh pig corneas followed by cDNA synthesis and analysis of VEGF and PDGF pathways by TaqMan Array gene expression profile. RESULTS: Normal pig cornea lacks VEGFR2 and VEGFR3 gene expression. MK2 and AKT1 genes were significantly overexpressed (p=0.000684, p=0.050995, respectively). Six PDGF pathway genes were overexpressed: TIAM1 (p=0.047), PIK3CA (p=0.00005), IKBKG (p=0.000006), PAK4 (p=0.034), RAC1 (p=0.000006 and PTGS2, p=0.00375). PDGF A was up-regulated, but not with a statistical significance (p=0.79911), while PDGFRα was down-regulated and PDGFRß was not expressed. CONCLUSION: Normal cornea avascularity is given by growth factor receptors down-regulation. Rapid corneal neovascularisation is induced by activation of the main angiogenic growth factors that induce angiogenic cascade and vessel recruitment.

Chloride Intracellular Channel Protein 1 (CLIC1), E-cadherin and P-cadherin Define Distinct Subclasses of HER2, Luminal B and Triple-negative Breast Cancer.

BACKGROUND/AIM: Chloride intracellular channel protein (CLIC1), E- and P-cadherin (Ecad, Pcad) are certified factors of aggressivity, but they have not been studied in breast cancer to date. The aim was to study CLIC1, Ecad and Pcad impact on breast cancer in terms of defining new high-risk subgroups. MATERIALS AND METHODS: Ninety-seven breast cancer biopsies were immunohistochemically evaluated for CLIC1, Ecad and Pcad expression related to molecular subtypes. CLIC1 expression was assessed in both tumor cells (CLIC1T) and blood vessels (CLIC1V). RESULTS: For 23% of Luminal A cases, both cadherins and CLIC1V were positive. Luminal B/HER2 subtype, had two specific phenotypes: Ecad-/Pcad-/CLIC1T-/CLIC1V+ and Ecad+/Pcad-/CLIC1T-/CLIC1V+. All TNBC cases were clustered into two subgroups: 60% were Ecad+/Pcad+/CLIC1T+/CLIC1V+) while 40% were Ecad+/Pcad+/CLIC1T+/CLIC1V-). CONCLUSION: CLIC1, Ecad and Pcad association stratifies molecular types of breast cancer in subgroups that may explain different response to therapy and different aggressiveness previously observed by other authors within the same molecular subtype.

Tumour-associated Angiogenesis and Intermediate Blood Vessels in Renal Cell Carcinoma.

Background/Aim: Renal cell carcinoma is strongly vascularized, and formation of new blood vessels is a complex and multi-step process. In this study, we analysed the subtypes of intermediate blood vessels, as shown by double immunohistochemistry. Materials and Methods: Tumour-associated blood vessels were identified by double immunostaining based on CD34 and smooth muscle cell actin. Blood vessels were classified both quantitatively and qualitatively based on the expression of the aforementioned two markers. The main criteria to sub-classify intermediate blood vessels was the presence, distribution, and arrangement of perivascular cells. Results: We described three subtypes of intermediate blood vessels found particularly in the tumour area: Subtype 1 lacked perivascular cells, subtype 2 showed scattered pericytes attached to the vascular wall, and subtype 3 showed a continuous layer of perivascular cells on one side. Conclusion: We describe for the first time three subtypes of renal cell carcinoma-associated intermediate blood vessels, which could be important in prognosis and as potential targets for anti-vascular therapy.

Historical Overview of In Vivo and In Vitro Angiogenesis Assays.

Most of angiogenesis assays were designed and developed during Folkman's era. But the growth of new blood vessels in several pathologic conditions as tumor development or inflammation were observed long time ago.The development of new blood vessels was early observed by ancient Egyptians who tried to destroy them by applying empirical methods. From the first observations regarding angiogenesis to a personalized therapy targeting newly formed blood vessels a lot of experimental in vitro and in vivo angiogenesis assays have been developed. The present work will overview the oldest and less known part of angiogenesis assays development, and in addition, it will present the newest data in the experimental field of angiogenesis which is rapidly improved by the needs of new antiangiogenic and antivascular therapy development.