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Introduction: Estrogens used in women’s healthcare have been associated with increased risks of venous thromboembolism (VTE) and breast cancer. Estetrol (E4), an estrogen produced by the human fetal liver, has recently been approved for the first time as a new estrogenic component of a novel combined oral contraceptive (E4/drospirenone [DRSP]) for over a decade. In phase 3 studies, E4/DRSP showed good contraceptive efficacy, a predictable bleeding pattern, and a favorable safety and tolerability profile. Areas covered: This narrative review discusses E4ʹs pharmacological characteristics, mode of action, and the results of preclinical and clinical studies for contraception, as well as for menopause and oncology. Expert opinion: Extensive studies have elucidated the properties of E4 that underlie its favorable safety profile. While classical estrogens (such as estradiol) exert their actions via both activation of nuclear and membrane estrogen receptor α (ERα), E4 presents a specific profile of ERα activation: E4 binds and activates nuclear ERα but does not induce the activation of membrane ERα signaling pathways in specific tissues. E4 has a small effect on normal breast tissue proliferation and minimally affects hepatic parameters. This distinct profile of ERα activation, uncoupling nuclear and membrane activation, is unique.

ObjectiveTo evaluate the bleeding patterns of a new combined oral contraceptive containing estetrol (E4) 15 mg/drospirenone (DRSP) 3 mg in a 24/4-day regimen.Study designWe pooled bleeding data from two parallel, open-label, 13-cycle phase 3 trials that enrolled participants 16 to 50 years old with body mass index (BMI) ≤35 kg/m2. Participants reported vaginal bleeding/spotting in daily diaries. For this bleeding analysis, we included participants with at least one evaluable cycle. We calculated mean frequencies of scheduled and unscheduled bleeding/spotting episodes and median duration of bleeding/spotting episodes, and assessed associations between treatment compliance, BMI and recent hormonal contraceptive use on bleeding/spotting outcomes.ResultsWe included 3409 participants with 33,815 cycles. Scheduled bleeding/spotting occurred in 87.2% to 90.4% of participants/cycle, with a median duration of 4 to 5 days. Unscheduled bleeding/spotting decreased from 27.1% in Cycle 1 to 20.6% in Cycle 2 to ≤17.5% from Cycle 5 onwards. Most (66.5%) unscheduled bleeding/spotting episodes were spotting-only. Between 5.8% and 7.8% of users/cycle experienced absence of any scheduled or unscheduled bleeding/spotting. Missing one or more active pills resulted in a higher occurrence of unscheduled bleeding/spotting (adjusted odds ratio [aOR] 2.13 [95% confidence interval 1.68-2.70]) and absence of scheduled bleeding/spotting (aOR 2.36 [1.82-3.07]). Participants with a BMI ≥30 kg/m2 reported more absence of scheduled bleeding/spotting (aOR 1.68 [1.37-2.05]). Switchers and starters reported similar frequencies of unscheduled bleeding/spotting (aOR 0.94 [0.83-1.07]) and absence of scheduled bleeding/spotting (aOR 1.00 [0.85-1.19]). Three percent of participants discontinued for a bleeding-related adverse event.ConclusionE4/DRSP use results in a predictable bleeding pattern with limited unscheduled bleeding/spotting. Noncompliance and BMI affect bleeding patterns.Implications statementMost estetrol/drospirenone users experience a predictable and regular bleeding pattern. Providers can educate patients about the expected bleeding patterns and should advise users that they may infrequently experience no scheduled bleeding/spotting. This information may improve user acceptability and continuation of this new oral contraceptive.

Objectives To evaluate the effect on endocrine and metabolic parameters of a new combined oral contraceptive (COC) containing estetrol (E4) and drospirenone (DRSP). Study design Randomized, open-label, controlled, 3-arm, parallel study. Healthy subjects received either E4 15 mg/DRSP 3 mg (E4/DRSP) (n = 38), or ethinylestradiol (EE) 30 µg/levonorgestrel (LNG) 150 µg (n = 29), or EE 20 µg/DRSP 3 mg (n = 31) for 6 treatment cycles. Median percentage change from baseline to cycle 3 and to cycle 6 were evaluated for endocrine parameters, liver proteins, lipid profile, and carbohydrate metabolism. Results At cycle 6, E4/DRSP treatment had less effect on gonadotropins (follicle stimulating hormone [FSH] +30.5%, luteinizing hormone [LH] −7.5%) compared to EE/LNG (FSH −84.0%, LH −92.0%) and EE/DRSP (FSH −64.0%, LH −90.0%). With E4/DRSP increases in total cortisol (+26.0%) and cortisol binding globulin ([CBG] (+40.0%) were less compared to EE/LNG (cortisol +109.0%, CBG +152.0%) and EE/DRSP (cortisol +107.0%, CBG +140.0%). Liver proteins, except CRP, increased, but the effect was less pronounced with E4/DRSP for angiotensinogen (+75.0%) compared to EE/LNG (+170.0%) and EE/DRSP (+206.5%) and for sex hormone binding globulin ([SHBG] +55.0%), compared to EE/LNG (+74.0%) and EE/DRSP (+251.0%). E4/DRSP had minimal impact on lipid parameters; the largest effect was observed for triglycerides (+24.0%), which was less compared to EE/LNG (+28.0%) and EE/DRSP (+65.5%). E4/DRSP had no effect on carbohydrate metabolism. Conclusions E4/DRSP treatment has limited effects on endocrine and metabolic parameters. The effects on gonadotropins, cortisol, CBG, angiotensinogen, SHBG and triglycerides were less pronounced compared to EE-containing products. Implications statement Combining E4 15 mg with DRSP 3 mg resulted in a COC with a different metabolic profile in comparison to EE-containing products. The clinical relevance of these findings needs to be further assessed, using clinical endpoints to establish the safety profile of this new COC.

Objective: To assess the effect on hemostasis parameters of a new combined oral contraceptive (COC). Study design: In this randomized, single centre, open-label, exploratory study, healthy women received either 15 mg estetrol/3 mg drospirenone (E4/DRSP) (n = 39), 30 mcg ethinylestradiol/150 mcg levonorgestrel (EE/LNG) (n = 30), or 20 mcg ethinylestradiol/3 mg drospirenone (EE/DRSP) (n = 32) for six 28day cycles. Blood was collected at baseline, cycle 3, and cycle 6. Median change from baseline was evaluated for procoagulant, anticoagulant, and fibrinolytic parameters, and for sex hormone-binding globulin (SHBG).Results: Median change of endogenous thrombin potential (ETP) based activated protein C sensitivity resistance (APCr) at cycle 6 was +30% for E4/DRSP, +165% for EE/LNG (p-value

Objective To evaluate the effects of estetrol 15 mg/drospirenone 3 mg on ovarian function. Study design Single-center, randomized, open-label, parallel study in healthy young women with proven ovulatory cycles. Participants received either estetrol 15 mg/drospirenone 3 mg (E4/DRSP) (n = 41) or ethinylestradiol 20 µg/drospirenone 3 mg (EE/DRSP) (n = 41) in a 24/4-day regimen for 3 consecutive cycles. Follicular size and endometrial thickness were measured by transvaginal ultrasound every 3 days in cycles 1 and 3. Blood was sampled for hormone analysis. Ovarian function expressed as Hoogland score was based on follicular size, serum estradiol (E2) and progesterone (P) concentrations. Ovulation was defined as a ruptured follicle-like structure >13 mm with serum E2 concentrations >100 pmol/L and serum P concentrations >5 nmol/L. We assessed return of ovulation after treatment cessation, and safety throughout the study. Results None of the participants ovulated with E4/DRSP use, while one participant ovulated once and one participant ovulated twice during EE/DRSP treatment. Most participants had a Hoogland score of 1 (no ovarian activity) in cycle 1 (85.0% and 82.9% of participants on E4/DRSP and EE/DRSP, respectively) and in cycle 3 (65.8% and 83.8%, respectively). E4/DRSP suppressed follicle-stimulating hormone and luteinizing hormone to a lesser extent than EE/DRSP, whereas both treatments comparably suppressed E2 and P and endometrial thickness. Return of ovulation occurred, on average, 15.5 days after E4/DRSP treatment discontinuation. E4/DRSP was safe and well-tolerated. Conclusions E4 15 mg/DRSP 3 mg results in adequate ovulation inhibition and ovarian function suppression, comparable to a marketed combined oral contraceptive containing EE/DRSP. Implications statement Treatment with E4 15 mg/DRSP 3 mg showed complete ovulation inhibition, despite less suppression of follicle-stimulating hormone and luteinizing hormone compared to EE/DRSP. If it becomes commercially available, E4/DRSP, containing a naturally occurring estrogen, should be as effective as EE/DRSP.

Objective The aim of this study was to select the minimum effective dose of estetrol (E4) for the treatment of vasomotor symptoms in postmenopausal women. Methods This was a multicenter, randomized, double-blind, placebo-controlled study. Postmenopausal women (n = 257, of whom 32 were hysterectomized) aged 40 to 65 years, with ≥7 moderate to severe hot flushes (HFs) per day, or 50 or more moderate to severe HFs weekly, received 2.5, 5, 10, or 15 mg E4, or placebo once-daily for a period of 12 weeks. Efficacy was assessed by recording the frequency and severity of HFs. Overall safety was assessed by recording adverse events, measuring endometrial thickness, and monitoring bleeding patterns. Treatment groups were compared using analysis of covariance. Results The frequency of moderate to severe HFs decreased with all E4 doses. The difference in the percentage change of weekly HF frequency was significant for 15 mg E4 versus placebo at both W4 (-66% vs -49%, P = 0.032) and W12 (-82% vs -65%, P = 0.022). The decrease in severity of HFs was significantly more pronounced for 15 mg E4 than for placebo at both W4 (-0.59 vs -0.33, P = 0.049) and W12 (-1.04 vs -0.66, P = 0.049); the other doses failed to achieve statistical significance. In nonhysterectomized women, endometrial thickness increased during treatment and normalized following progestin treatment at study completion. No endometrial hyperplasia was observed. Conclusions Estetrol 15 mg is considered to be the minimum effective daily oral dose for treatment of vasomotor symptoms. Its current seemingly favorable safety profile is further to be confirmed in phase 3 clinical development. : Video Summary:http://links.lww.com/MENO/A591.

Background: Most combined oral contraceptives (COCs) contain ethinylestradiol (EE), an estrogen component known to increase the risk of venous thromboembolic events. Previous phase 2 trials have shown that Estetrol (E4), a naturally-occurring estrogen produced by the human fetal liver, in combination with Drospirenone (DRSP), inhibits ovulation and is associated with favorable vaginal bleeding, safety and tolerability profiles and high user satisfaction. When administered in doses up to 10 mg for less than 3 months, E4 either alone or in combination with DRSP showed limited effects on liver function as well as on metabolic and endocrine parameters. However, the impact of E4 15 mg/DRSP 3 mg, the selected dose for pregnancy prevention (hereafter referred to as E4/DRSP), on metabolic and endocrine parameters was never investigated. Methods: A randomized, open-label, controlled, three-arm parallel study was conducted in 101 healthy volunteers aged 18-50 years with a body mass index between 18.0 and 30.0 kg/m2. Of the randomized individuals, 98 subjects received either E4/DRSP (n=38), EE 30 µg/levonorgestrel (LNG) 150 µg (n=29) or EE 20 µg/DRSP 3 mg (n=31) COCs for six consecutive treatment cycles of 28 days. Endocrine and metabolic parameters were measured at baseline, cycle 3 and cycle 6. Results: FSH and LH decreased with both EE/LNG (-84% and -92%, respectively) and EE/DRSP (-64% and -90%, respectively), whereas E4/DRSP slightly increased FSH (31%) and had minor effect on LH (-8%). Total cortisol increased by more than 100% during treatment with EE/LNG (109%) and EE/DRSP (107%), while E4/DRSP only slightly increased total cortisol (26%). The effects of E4/DRSP, EE/LNG and EE/DRSP on other endocrine parameters including androstenedione (-31%, -49% and -40%) and free testosterone (-50%, -50% and -71%) were similar. Liver proteins, except CRP, increased from baseline in all treatment groups. The increase in angiotensinogen, CBG, and TBG levels was significantly lower for E4/DRSP (75%, 40% and 17%) in comparison with EE/LNG (170%, 152% and 37%) and EE/DRSP (207%, 140% and 70%). SHBG substantially increased for EE/DRSP (251%), while EE/LNG and E4/DRSP had limited effects on SHBG (74% and 55%, respectively). Triglycerides changed to a lesser extent for E4/DRSP (24%) compared to EE/DRSP (66%); EE/LNG increased triglycerides levels by 28%. Moreover, E4/DRSP had minimal impact on lipid parameters including HDL (4%), Apolipoprotein A1 (5%) and Apolipoprotein B (4%) compared to baseline, and no effects on carbohydrate metabolism. Conclusion: These data confirm that E4/DRSP has limited effects on liver proteins, lipid and carbohydrate metabolism, cortisol, and gonadotropins. The effect on other endocrine parameters, including suppression of ovarian steroids, was typical for a COC. In conclusion, combining E4 15 mg with DRSP 3 mg resulted in a different and potentially favorable metabolic profile.

Estetrol (E4) acts as a typical estrogen (E) agonist in stimulating the endometrium, and when administered to postmenopausal women in various doses, reverses vaginal atrophy and decreases vasomotor symptoms. However, in contrast to other Es administered orally, E4 demonstrates a minimal hepatic effect and does not stimulate breast proliferation. Data in pre and postmenopausal women show minimal stimulatory effects on triglycerides, sex hormone binding globulin, corticosteroid-binding globulin, and angiotensinogen. Similarly, E4 exerts minimal effects on coagulation markers, and when combined with drospirenone as a combined oral contraceptive does not increase the generation of thrombin as measured by the activated protein C resistance ratio, unlike the use of ethinylestradiol. Because of the tissue selectivity of E4, this study sought to better delineate the molecular mode of action of E4. Selective E Receptor Modulators (SERMs) exhibit specific cell and tissue estrogenic, neutral or anti-estrogenic activities. SERMs interact with the ligand binding domain of the E Receptor alpha (ERα) in a way that is distinct from that of E. By preventing the kinking of helix 12 in ERα, SERMs recruit other co-receptor activators and inhibitors that are responsible for SERM action. We compared in replicates the binding pattern and affinities of 154 motifs of ERα co-regulator activators and repressors to complexes of ERα with Estradiol (E2), Estriol (E3) or E4 in a specific Pam Gene assay (1).The pattern of co-receptor binding in the presence of E4 was identical to that elicited by E2 and E3, but with a 50-fold lower potency. This pattern is strikingly distinct from that observed with 4-hydroxytamoxifen or raloxifen. These data together with previously published studies of the crystalline structures of ERα with E2, E3 or E4 complexes confirm that E4 interacts with nuclear ERα in a manner identical to that of the other Es (2). Previous studies have shown that E4 inhibits the activity of the membrane ERα, and blocks the stimulatory action of E2 in this membrane ERα subpopulation. In conclusion, E4 is a unique E with specific actions in tissues, demonstrating absence of specific membrane receptor effects, and no interaction with ERα in the way characteristic of SERMs. The unique profile of E4 activity with lower hepatic effects (minimal or no effects on triglycerides and coagulation) may ultimately translate into safer clinical use. 1) Broekema et al. Endocrinology. 2018; 159:2397. 2) Abot et al. EMBO Mol Med. 2014; 6:1328 Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Estrogens (E), in oral contraceptives (COCs) and hormone replacement therapy (HRT) drugs used for the relief of climacteric symptoms of menopause, increase the synthesis of clotting factors, decrease the levels of coagulation inhibitors, and increase the risk of venous thromboembolic events (VTE). Ischemic stroke incidence in postmenopausal women during HRT use is also increased and is probably due to a thrombotic event. This suggests that a safer estrogen may reduce stroke and VTE incidence, with lower impact on hemostasis.

Objectives The primary objective was to evaluate the safety and local tolerance of a topical 2% (w/w) cidofovir gel, applied directly to the cervices of women with high-grade cervical intraepithelial neoplasia (CIN 2+). The secondary objective was to evaluate the pharmacokinetics of cidofovir during the treatment. Materials and methods Nine women with CIN 2+, were treated with a course of 3 g of cidofovir gel, applied locally once per week for 3 weeks in total (9 g). The treatment was administered in a cervical cap, applied to the cervix for 5 or 10 hours (n = 6 and 3 patients, respectively). Follow-up included a structured questionnaire, a gynecological examination, blood analysis for hematology, C-reactive protein (CRP), and renal function assessment plus pharmacokinetic analyses of cidofovir after each treatment and at the end of the full course. Results No clinically significant hematological/biochemical abnormalities or serious adverse events (SAE) were reported, although 6 mild to moderate adverse events (AE) occurred in relation to the study drug: 1 flu-like syndrome and 5 local AEs. Plasma concentrations of cidofovir were very low (mean Cmax of 103.0 and 99.2 ng/mL after 5 and 10 hours of exposure, respectively). Conclusion Cidofovir, directly applied on CIN 2+, is reasonably well tolerated and the systemic exposure following topical application is much lower than that seen with intravenous administration, at the approved dose. .

Author(s): Creinin, Mitchell D; Rosing, Jan; Jost, Maud; Kinet, Virginie; Chatel, Guillaume; Foidart, Jean Michel

An adequate balance between serine proteases and their plasminogen activator inhibitor-1 (PAI-1) is critical for pathological angiogenesis. PAI-1 deficiency in mice is associated with impaired choroidal neovascularization (CNV) and tumoral angiogenesis. In the present work, we demonstrate unexpected differences in the contribution of bone marrow (BM)-derived cells in these two processes regulated by PAI-1. PAI-1−/− mice grafted with BM-derived from wild-type mice were able to support laser-induced CNV formation but not skin carcinoma vascularization. Engraftment of irradiated wild-type mice with PAI-1−/− BM prevented CNV formation, demonstrating the crucial role of PAI-1 delivered by BM-derived cells. In contrast, the transient infiltration of tumor transplants by local PAI-1-producing host cells rather than by BM cells was sufficient to rescue tumor growth and angiogenesis in PAI-1-deficient mice. These data identify PAI-1 as a molecular determinant of a local permissive soil for tumor angiogenesis. Altogether, the present study demonstrates that different cellular mechanisms contribute to PAI-1-regulated tumoral and CNV. PAI-1 contributes to BM-dependent choroidal vascularization and to BM-independent tumor growth and angiogenesis.

Objectives The aim of the study was to evaluate the efficacy of different dosages of estetrol (E4) combined with one of two progestins in suppressing the pituitary–ovarian axis and ovulation in healthy premenopausal women. Methods This was an open, parallel, phase II, dose-finding, pilot study performed in healthy women aged 18 to 35 years with a documented ovulatory cycle before treatment. For three consecutive cycles in a 24/4-day regimen, participants received 5 mg or 10 mg E4/3 mg drospirenone (DRSP); 5 mg, 10 mg or 20 mg E4/150 μg levonorgestrel; or 20 μg ethinylestradiol (EE)/3 mg DRSP as comparator. Pituitary–ovarian axis activity and the occurrence of ovulation were evaluated by monitoring follicular size, serum levels of follicle-stimulating hormone, luteinising hormone, estradiol and progesterone during treatment cycles 1 and 3. Endometrial thickness was evaluated throughout the trial, and the return of ovulation was evaluated after the last intake of medication. Results A total of 109 women were included in the trial. No ovulation occurred in any treatment group. Ovarian activity inhibition seemed proportional to the E4 dosage: the highest suppression was observed in the 20 mg E4 group and was very similar to that observed with EE/DRSP. Endometrial thickness was suppressed to the same extent in all groups. Post-treatment ovulation occurred in all participants between 17 and 21 days after the last active treatment. The study combinations were well tolerated and safe. Conclusions Combined with a progestin, E4 adequately suppresses ovarian activity, particularly when given at a dosage above 10 mg/day. Chinese Abstract 摘要 目的 这项研究的目的是评估不同剂量的雌四醇联合两种孕激素中的其中一种对垂体-卵巢轴以及健康的绝经前妇女的排卵方面的抑制疗效。 方法 这是一个在18到35岁的健康女性中进行的开放的、平行的，关于II期药物剂量探索的初步研究，这些女性均有治疗前的排卵周期记录。连续三个周期的24/4-天方案,参与者接受5毫克或10毫克的雌四醇/3毫克屈螺酮;5毫克,10毫克或20毫克的雌四醇/150 μg左炔诺孕酮;或用20 μg炔雌醇/3毫克屈螺酮作比较。在第1和第3个治疗周期，通过监测卵泡的大小,血清促卵泡激素、黄体生成素、雌二醇和孕激素的水平对垂体-卵巢轴的活动和排卵进行评估。对子宫内膜厚度的评估贯穿于整个试验过程中，对排卵抑制的评估在用完药物之后。 结果 总共有109名妇女参与试验。任何一个治疗组都没有出现排卵。对卵巢活动的抑制似乎与雌四醇的剂量成正比：最严重的抑制出现在20毫克的雌四醇组，与在炔雌醇/屈螺酮组观察到的情况非常相似。所有组的子宫内膜厚度的抑制程度是相同的。在最后一次积极治疗后的第17到21天之间，所有参与者均出现了治疗后的排卵。所有的试验组合均有良好的耐受性和安全性。 结论 雌四醇联合一种雌激素能够充分的抑制卵巢活动，尤其是当雌四醇的剂量大于10毫克/天的时候。 关键词 雌四醇；雌激素；口服避孕药；排卵抑制；孕激素

The matrix metalloproteinases (MMPs) play a key role in normal and pathological angiogenesis by mediating extracellular matrix degradation and/or controlling the biological activity of growth factors, chemokines, and/or cytokines. Specific functions of individual MMPs as anti- or proangiogenic mediators remain to be elucidated. In the present study, we assessed the impact of single or combined MMP deficiencies in in vivo and in vitro models of angiogenesis (malignant keratinocyte transplantation and the aortic ring assay, respectively). MMP-9 was predominantly expressed by neutrophils in tumor transplants, whereas MMP-2 and MMP-3 were stromal. Neither the single deficiency of MMP-2, MMP-3, or MMP-9, nor the combined absence of MMP-9 and MMP-3 did impair tumor invasion and vascularization in vivo. However, there was a striking cooperative effect in double MMP-2:MMP-9-deficient mice as demonstrated by the absence of tumor vascularization and invasion. In contrast, the combined lack of MMP-2 and MMP-9 did not impair the in vitro capillary outgrowth from aortic rings. These results point to the importance of a cross talk between several host cells for the in vivo tumor promoting and angiogenic effects of MMP-2 and MMP-9. Our data demonstrate for the first time in an experimental model that MMP-2 and MMP-9 cooperate in promoting the in vivo invasive and angiogenic phenotype of malignant keratinocytes.

Plasminogen activator inhibitor type 1 (PAI-1) plays a key role in tumor progression and is believed to control proteolytic activity and cell migration during angiogenesis. We report here that host PAI-1, at physiological concentration, promotes in vivo tumor invasion and angiogenesis. In sharp contrast, inhibition of tumor vascularization was observed when PAI-1 was produced at supraphysiologic levels, either by host cells (transgenic mice overexpressing PAI-1) or by tumor cells (after transfection with murine PAI-1 cDNA). This study provides for the first time in vivo evidence for a dose-dependent effect of PAI-1 on tumor angiogenesis. Of great interest is the finding that PAI-1 produced by tumor cells, even at high concentration, did not overcome the absence of PAI-1 in the host, emphasizing the importance of the cellular source of PAI-1.

New blood formation or angiogenesis has become a key target in therapeutic strategies aimed at inhibiting tumor growth and other diseases associated with neovascularization. Angiogenesis is associated with important extracellular remodeling involving different proteolytic systems among which the plasminogen system plays an essential role. It belongs to the large serine proteinase family and can act directly or indirectly by activating matrix metalloproteinases or by liberating growth factors and cytokines sequestered within the extracellular matrix. Migration of endothelial cells is associated with significant upregulation of proteolysis and, conversely, immunoneutralization or chemical inhibition of the system reduces angiogenesis in vitro. On the other hand, genetically altered mice developed normally without overt vascular anomalies indicating the possibility of compensation by other proteases in vivo. Nevertheless, they have in some experimental settings revealed unanticipated roles for previously characterized proteinases or their inhibitors. In this review, the complex mechanisms of action of the serine proteases in pathological angiogenesis are summarized alongside possible therapeutic applications.

The tumor is a complex system comprising neoplastic genetically altered cells and a tumor stroma composed of remodeled extracellular matrix, newly formed vessels, and infiltrating host cells. The development of a cancer is a progressive multistep process in which neoplastic cells progress to malignancy by activating their microenvironment and by responding to the tumor-supporting cues of the surrounding tissue. Because of the recently recognized importance of a permissive stroma for tumor development and invasion, the host compartment is now viewed as an interesting new target for tumor therapy. Among positive regulators contributing to the elaboration of this permissive stroma are growth factors, cytokines/chemokines, proteases, and their inhibitors. The present review summarizes what we learned during the last decade on the contribution of these factors at the tumor–host interface by exploiting a useful in vivo surface transplantation model of skin carcinomas.

An adequate balance between serine proteases and their plasminogen activator inhibitor-1 (PAI-1) is critical for pathological angiogenesis. PAI-1 deficiency in mice is associated with impaired choroidal neovascularization (CNV) and tumoral angiogenesis. In the present work, we demonstrate unexpected differences in the contribution of bone marrow (BM)-derived cells in these two processes regulated by PAI-1. PAI-1(-/-) mice grafted with BM-derived from wild-type mice were able to support laser-induced CNV formation but not skin carcinoma vascularization. Engraftment of irradiated wild-type mice with PAI-1(-/-) BM prevented CNV formation, demonstrating the crucial role of PAI-1 delivered by BM-derived cells. In contrast, the transient infiltration of tumor transplants by local PAI-1-producing host cells rather than by BM cells was sufficient to rescue tumor growth and angiogenesis in PAI-1-deficient mice. These data identify PAI-1 as a molecular determinant of a local permissive soil for tumor angiogenesis. Altogether, the present study demonstrates that different cellular mechanisms contribute to PAI-1-regulated tumoral and CNV. PAI-1 contributes to BM-dependent choroidal vascularization and to BM-independent tumor growth and angiogenesis.

The matrix-inserted surface transplantation model is an in vivo assay used to analyse the kinetics of tumor-vessel interactions during different stages of skin carcinoma progression. This system allows the study of host-tumor interface, i.e. penetration of tumor cells into normal host tissue as well as infiltration of normal host cells into the tumor. In the present study, image analysis algorithms for processing and quantifying the extent of such migratory and tissue remodeling events are presented. The proposed method is non-parametric and its originality lies in its particularity to take into account the specific geometry of tumor-host interface. This methodology is validated by evaluating the contribution of matrix metalloproteases (MMPs) in skin carcinoma invasion and vascularization through pharmacological and genetic approaches.

The increasing diversity in both substrates and functions of matrix metalloproteinases (MMPs) makes these enzymes central regulators in the complex tumor ecosystem composed of cancer cells and their microenvironment. In the majority of cancers, membrane-associated and extracellular proteases are mainly produced by host cells including inflammatory cells, endothelial cells, pericytes and fibroblasts. Recent data based on in vitro and in vivo studies have demonstrated the relevance of these enzymes in multiple processes controlling cancer growth, angiogenesis and metastatic dissemination. This review will present the emerging MMP-related features of cancer cells and host cells.

Age-related macular degeneration (AMD) is the leading cause of blindness in elderly patients. The more aggressive exudative form is characterized by abnormal blood-vessel development that occurs beneath the retina as a result of choroidal neovascularization (CNV). Vascular endothelial growth factor (VEGF) has emerged as the key mediator of CNV formation; this has led to intensive research on VEGF and the recent approval of anti-VEGF compounds by the US Food and Drug Administration. Despite this successful introduction of anti-angiogenic therapies into the clinical setting, there is still a lack of treatments that definitively reverse damaged vision. Here, we consider the importance of putative molecular targets other than VEGF that might have been underestimated. Emerging cellular mechanisms offer additional opportunities for innovative therapeutic approaches.

Among matrix metalloproteinases (MMP), MMP-19 displays unique structural features and tissue distribution. In contrast to most MMPs, MMP-19 is expressed in normal human epidermis and down-regulated during malignant transformation and dedifferentiation. The contribution of MMP-19 during tumor angiogenesis is presently unknown. In an attempt to give new insights into MMP-19 in vivo functions, angiogenic response of mutant mice lacking MMP-19 was analyzed after transplantation of murine malignant PDVA keratinocytes and after injection of Matrigel supplemented with basic fibroblast growth factor. In situ hybridization and immunohistochemical analysis revealed that MMP-19 is produced by host mesenchymal cells but not by endothelial capillary cells or CD11b-positive inflammatory cells. Based on a new computer-assisted method of quantification, we provide evidence that host MMP-19 deficiency was associated with an increased early angiogenic response. In addition, increased tumor invasion was observed in MMP-19−/− mice. We conclude that, in contrast to most MMPs that promote tumor progression, MMP-19 is a negative regulator of early steps of tumor angiogenesis and invasion. These data highlight the requirement to understand the individual functions of each MMP to improve anticancer strategies. (Cancer Res 2006; 66(10): 5234-41)

Angiogenesis and tumor expansion are associated with extracellular matrix remodeling and involve various proteases such as the plasminogen (Pig)/plasminogen activator (PA) system. Recently, several experimental data have implicated the plasminogen activator inhibitor-1 (PAI-1) in tumor angiogenesis in murine systems. However, little is known about PAI-1 functions in human skin carcinoma progression. By generating immunodeficient mice (in Rag-1-/- or nude background) deleted for PAI-1 gene (PAI-1-/- ), we have evaluated the impact of host PAI-1 deficiency on the tumorigenicity of two malignant human skin keratinocyte cell lines HaCaT II-4 and HaCaT A5-RT3 forming low-grade and high-grade carcinomas, respectively. When using the surface transplantation model, angiogenesis and tumor invasion of these two cell lines are strongly reduced in PAI-1-deficient mice as compared to the wild-type control animals. After subcutaneous injection in PAI-1-/- mice, the tumor incidence is reduced for HaCaT II-4 cells, but not for those formed by HaCaT A5-RT3 cells. These data indicate that PAI-1 produced by host cells is an important contributor to earlier stages of human skin carcinoma progression. It exerts its tumor-promoting effect in a tumor stage-dependent manner, but PAI-1 deficiency is not sufficient to prevent neoplastic growth of aggressive tumors of the human skin.

Cell surface proteolysis is an important mechanism for generating biologically active proteins that mediate a range of cellular functions and contribute to biological processes such as angiogenesis. Although most studies have focused on the plasminogen system and matrix metalloproteinases (MMPs), recently there has been an increase in the identification of membrane associated proteases, including serine proteases, ADAMs, and membrane-type MMPs (MT-MMPs). Normally, protease activity is tightly controlled by tissue inhibitors of MMPs (TIMPs) and plasminogen activator inhibitors (PAIs). The balance between active proteases and inhibitors is thought to determine the occurrence of proteolysis in vivo. High concentrations of proteolytic system components correlate with poor prognosis in many cancers. Paradoxically, high (not low) PAI-1 or TIMP concentrations predict poor survival in patients with various cancers. Recent observations indicate a much more complex role for protease inhibitors in tumour progression and angiogenesis than initially expected. As knowledge in the field of protease biology has improved, the unforeseen complexities of cell associated enzymes and their interaction with physiological inhibitors have emerged, often revealing unexpected mechanisms of action.

Matrix metalloproteinase 2 (MMP-2) and MMP-9 are increased in human choroidal neovascularization (CNV) occurring during the exudative most aggressive form of age-related macular degeneration (AMD), but their precise role and potential interactions remain unclear. To address the question of MMP-2 and MMP-9 functions, mice deficient in the expression of MMP-2 (MMP-2 KO), MMP-9 (MMP-9 KO), and both MMP-2 and MMP-9 (MMP-2,9 KO) with their corresponding wild-type mice (WT) underwent CNV induction by laser-induced rupture of the Bruch's membrane. Both the incidence and the severity of CNV were strongly attenuated in double deficient compared with single gene deficient mice or corresponding WT controls. The reduced neovascularization was accompanied by fibrinogen/fibrin accumulation. Furthermore, overexpression of the endogenous MMP inhibitors TIMP-1 or TIMP-2 (delivered by adenoviral vectors) in WT mice or daily injection of a synthetic and gelatinase selective MMP inhibitor (Ro 26-2853) significantly decreased the pathological reaction. These findings suggest that MMP-2 and MMP-9 may cooperate in the development of AMD and that their selective inhibition represents an alternative strategy for the treatment of choroidal neovascularization.

Age-related macular degeneration (AMD) is the primary cause of irreversible photoreceptors loss in adult patients and current therapies are limited. Increased levels of matrix metalloproteinases (MMPs) have been documented in neovascularization of severe ocular pathologies such as AMD and proliferative diabetic retinopathy. We report here that MMP-9 (gelatinase B) expression is induced and temporally regulated in the course of experimental choroidal neovascularization. We used transgenic mice expressing beta-galactosidase reporter gene under the dependence of MMP-9 promoter and RT-PCR analysis on choroidal neovascular structures microdissected from serial sections by laser pressure catapulting to show that MMP-9 expression is up-regulated concomitantly with the appearance of inflammatory cells in the subretinal lesion. In mice deficient in MMP-9 expression the development of choroidal neovascularization induced by laser photocoagulation still occurred, but at a reduced level.

Plasminogen activator inhibitor-1 is known to play a paradoxical positive role in tumor angiogenesis, but its contribution to metastatic spread remains unclear. We studied the impact of plasminogen activator inhibitor (PAI)-1 deficiency in a transgenic mouse model of ocular tumors originating from retinal epithelial cells and leading to brain metastasis (TRP-1/SV40 Tag mice). PAI-1 deficiency did not affect primary tumor growth or vascularization, but was associated with a smaller number of brain metastases. Brain metastases were found to be differentially distributed between the two genotypes. PAI-1-deficient mice displayed mostly secondary foci expanding from local optic nerve infiltration, whereas wild-type animals displayed more disseminated nodules in the scissura and meningeal spaces. SuperArray GEarray analyses aimed at detecting molecules potentially compensating for PAI-1 deficiency demonstrated an increase in fibroblast growth factor-1 (FGF-1) gene expression in primary tumors, which was confirmed by reverse transcription-polymerase chain reaction and western blotting. Our data provide the first evidence of a key role for PAI-1 in a spontaneous model of metastasis and suggest that angiogenic factors, such as FGF-1, may be important for primary tumor growth and may compensate for the absence of PAI-1. They identify PAI-1 and FGF-1 as important targets for combined antitumor strategies.