Aldehyde dehydrogenase 1A1 (ALDH1A1), a member of aldehyde dehydrogenase family, is a marker of stemness in breast cancer. During tumor progression cancer stem cells (CSCs) have been reported to secrete angiogenic factors to orchestrate the formation of pathological angiogenesis. This vasculature can represent the source of self-renewal of CSCs and the route for further tumor spreading. The aim of the present study has been to assess whether ALDH1A1 controls the output of angiogenic factors in breast cancer cells and regulates tumor angiogenesis in a panel of in vitro and in vivo models. Stemness status of breast cancer cells was evaluated by the ability to form turmorspheres in vitro. A transwell system was used to assess the angiogenic features of human umbilical vein endothelial cells (HUVEC) when co-cultured with breast cancer cells MCF-7 harboring different levels of ALDH1A1. Under these conditions, we survey endothelial proliferation, migration, tube formation and permeability. Moreover, in vivo, MCF-7 xenografts in immunodeficient mice allow to evaluate blood flow, expression of angiogenic factors and microvascular density (MVD). In MCF-7 we observed that ALDH1A1 activity conferred stemness property and its expression correlated with an activation of angiogenic factors. In particular we observed a significant upregulation of hypoxia inducible factor-1α (HIF-1α) and proangiogenic factors, such as vascular endothelial growth factor (VEGF). High levels of ALDH1A1, through the retinoic acid pathway, were significantly associated with VEGF-mediated angiogenesis in vitro. Co-culture of HUVEC with ALDH1A1 expressing tumor cells promoted endothelial proliferation, migration, tube formation and permeability. Conversely, downregulation of ALDH1A1 in MCF-7 resulted in reduction of proangiogenic factor release/expression and impaired HUVEC angiogenic functions. In vivo, when subcutaneously implanted in immunodeficient mice, ALDH1A1 overexpressing breast tumor cells displayed a higher expression of VEGF and MVD. In breast tumors, ALDH1A1 expression primes a permissive microenvironment by promoting tumor angiogenesis via retinoic acid dependent mechanism. In conclusion, ALDH1A1 might be associated to progression and diffusion of breast cancer.

Melanoma and non-small-cell lung carcinoma (NSCLC) cell lines are characterized by an intrinsic population of cancer stem-like cells (CSC), and high expression of detoxifying isozymes, the aldehyde dehydrogenases (ALDHs), regulating the redox state. In this study, using melanoma and NSCLC cells, we demonstrate that ALDH3A1 isozyme overexpression and activity is closely associated with a highly aggressive mesenchymal and immunosuppressive profile. The contribution of ALDH3A1 to the stemness and immunogenic status of melanoma and NSCLC cells was evaluated by their ability to grow in 3D forming tumorspheres, and by the expression of markers for stemness, epithelial to mesenchymal transition (EMT), and inflammation. Furthermore, in specimens from melanoma and NSCLC patients, we investigated the expression of ALDH3A1, PD-L1, and cyclooxygenase-2 (COX-2) by immunohistochemistry. We show that cells engineered to overexpress the ALDH3A1 enzyme enriched the CSCs population in melanoma and NSCLC cultures, changing their transcriptome. In fact, we found increased expression of EMT markers, such as vimentin, fibronectin, and Zeb1, and of pro-inflammatory and immunosuppressive mediators, such as NFkB, prostaglandin E2, and interleukin-6 and -13. ALDH3A1 overexpression enhanced PD-L1 output in tumor cells and resulted in reduced proliferation of peripheral blood mononuclear cells when co-cultured with tumor cells. Furthermore, in tumor specimens from melanoma and NSCLC patients, ALDH3A1 expression was invariably correlated with PD-L1 and the pro-inflammatory marker COX-2. These findings link ALDH3A1 expression to tumor stemness, EMT and PD-L1 expression, and suggest that aldehyde detoxification is a redox metabolic pathway that tunes the immunological output of tumors.

Carbonic anhydrase IX (CA-IX) plays a pivotal role in regulation of pH in tumor milieu catalyzing carbonic acid formation by hydrating CO₂. An acidification of tumor microenvironment contributes to tumor progression via multiple processes, including reduced cell-cell adhesion, increased migration and matrix invasion. We aimed to assess whether the pharmacological inhibition of CA-IX could impair tumor cell proliferation and invasion. Tumor epithelial cells from breast (MDA-MB-231) and lung (A549) cancer were used to evaluate the cytotoxic effect of sulfonamide CA-IX inhibitors. Two CA-IX enzyme blockers were tested, SLC-0111 (at present in phase Ib clinical trial) and AA-06-05. In these cells, the drugs inhibited cell proliferation, migration and invasion through shifting of the mesenchymal phenotype toward an epithelial one and by impairing matrix metalloprotease-2 (MMP-2) activity. The antitumor activity was elicited via apoptosis pathway activation. An upregulation of p53 was observed, which in turn regulated the activation of caspase-3. Inhibition of proteolytic activity was accompanied by upregulation of the endogenous tissue inhibitor TIMP-2. Collectively, these data confirm the potential use of CA-IX inhibitors, and in particular SLC-0111 and AA-06-05, as agents to be further developed, alone or in combination with other conventional anticancer drugs.

Hyaluronic acid (HA) is used in substitutive and aesthetic medicine with various applications. Ultrapure absorbable HA (Bioregen^®) and a mix of reticulated and free low molecular weight HA (Regenyal Idea Bioexpander^®) (both provided by Regenyal Laboratories Srl, San Benedetto del Tronto (AP), Italy) represent a reliable hydrating device and skin filler, useful for skin blemishes, lines and wrinkles, and lip widening, respectively. The commercial products are known for their safety, but data on the molecular, cellular, and tissue responses are lacking. We aimed to evaluate the bioavailability and the pro-angiogenic features of the products Bioregen^® and Bioexpander^® in vitro on cultured endothelial cells (ECs) and dermal fibroblasts in vivo when injected into experimental animals. When added to fibroblasts and ECs, Bioexpander^® induced cell migration. The two HA preparations were well tolerated, while a transient proangiogenic behavior of Bioexpander^®, when implanted subcutaneously in mice, was found. The neovascular response was evident in the first week with higher levels of VEGF and FGF-2 before undergoing regression. In conclusion, our data strengthen the safety of HA synthetic preparations both in vitro and in vivo. Even if a proangiogenic response is documented, it is modest and transient, leading to tissue recovery and absence of an inflammatory infiltrate.

The vascular endothelium consists of a single layer of squamous endothelial cells (ECs) lining the inner surface of blood vessels. Nowadays, it is no longer considered as a simple barrier between the blood and vessel wall, but a central hub to control blood flow homeostasis and fulfill tissue metabolic demands by furnishing oxygen and nutrients. The endothelium regulates the proper functioning of vessels and microcirculation, in terms of tone control, blood fluidity, and fine tuning of inflammatory and redox reactions within the vessel wall and in surrounding tissues. This multiplicity of effects is due to the ability of ECs to produce, process, and release key modulators. Among these, gasotransmitters such as nitric oxide (NO) and hydrogen sulfide (H₂S) are very active molecules constitutively produced by endotheliocytes for the maintenance and control of vascular physiological functions, while their impairment is responsible for endothelial dysfunction and cardiovascular disorders such as hypertension, atherosclerosis, and impaired wound healing and vascularization due to diabetes, infections, and ischemia. Upregulation of H₂S producing enzymes and administration of H₂S donors can be considered as innovative therapeutic approaches to improve EC biology and function, to revert endothelial dysfunction or to prevent cardiovascular disease progression. This review will focus on the beneficial autocrine/paracrine properties of H₂S on ECs and the state of the art on H₂S potentiating drugs and tools.

Preservation of vascular wall integrity against degenerative processes associated with ageing, fat-rich diet and metabolic diseases is a timely therapeutical challenge. The loss of endothelial function and integrity leads to cardiovascular diseases and multiorgan inflammation. The protective effects of the H₂S-donor erucin, an isothiocyanate purified by Eruca sativa Mill. seeds, were evaluated on human endothelial and vascular smooth muscle cells. In particular, erucin actions were evaluated on cell viability, ROS, caspase 3/7, inflammatory markers levels and the endothelial hyperpermeability in an inflammatory model associated with high glucose concentrations (25 mM, HG). Erucin significantly prevented the HG-induced decrease in cell viability as well as the increase in ROS, caspase 3/7 activation, and TNF-α and IL-6 levels. Similarly, erucin suppressed COX-2 and NF-κB upregulation associated with HG exposure. Erucin also caused a significant inhibition of p22phox subunit expression in endothelial cells. In addition, erucin significantly prevented the HG-induced increase in endothelial permeability as also confirmed by the quantification of the specific markers VE-Cadherin and ZO-1. In conclusion, our results assess anti-inflammatory and antioxidant effects by erucin in vascular cells undergoing HG-induced inflammation and this protection parallels the preservation of endothelial barrier properties.