Vascular Obliteration Due To Endothelial And Myointimal Growth In COVID-19

Background: Severe coronavirus disease 2019 (Covid-19) is a systemic multi-organ viral invasion. Previous studies found that many patients had a procoagulant state and/or severe hypoxemia with relatively well-preserved lung mechanics. Mechanisms underlying the vascular and its surrounding tissue are not well known yet. Histological data in Covid-19 tissues´ patients are still limited and mainly focused on post-mortem analysis. Since SARS-CoV-2 largely affects cutaneous tissue, we aim to assess the pathophysiologic mechanisms in depth in living skin tissue related to Covid-19. Methods: Five skin lesions from caucasian Covid-19 adult patients were selected for cutaneous tissue histological examination including immunohistochemistry (IHC) and direct immunouorescent (DIF) vast amount of data. Results: A common strong vasculopathic reaction pattern based on prominent vascular endothelial and myointimal cellgrowth was identied. Endothelial cell distortion generated vascular lumen obliteration and a strike erythrocyte and serum extravasation. Extensive signicant vascular C4d and C3 deposition throughout vascular cell wall was also identied. A regenerative epidermal hyperplasia with tissue structure preservation was also found. Conclusions: Covid-19 could comprise an obliterative micro-angiopathy consisting on endothelial and myointimal intensive growth with complement activation. This mechanism, together with increased vascular permeability identied, could contribute to obliterative vascular lumen and hemorrhage in Covid-19 disease. Activation of the complement and angiogenic pathways could have an important role in inducing and maintaining this vasculopathic reaction pattern. Thus, anticoagulation by itself could not completely reverse vascular lumen obliteration, with consequent hemorrhagic increased risk associated. Skin is the largest organ in the body, the most accessible one and can mirror other organs of the body. Findings of this study could contribute to a better understanding of physio-pathological mechanisms underlying Covid-19 infection on living tissue and could help further studies nd potential targets for specic therapeutic interventions in Covid-19 severe patients. (Agilent Dako Coverstainer, Glostrup, Denmark, SN-CS100-10390), Iron stain (Agilent Dako Artisan, Glostrup, Denmark, SN-ALP913100), and Periodic Acid Schiff (PAS) (Agilent Dako Artisan, Glostrup, Denmark, SN-ALP913100) staining were evaluated. Briey, density and composition of inammatory cells, the presence of obliterative proliferative endothelitis, vascular thrombosis, recent hemorrhage (extravascular erythrocyte cells), hemosiderin and brin deposits were assessed. The histological study was completed with IHC and IFD techniques. IHC for CD31, Smooth-muscle Actin (SMA), Ki67, Cyclin D1 and C4d was performed on formalin-xed, paran-embedded tissues using standard streptavidin-biotin peroxidase detection system (automated staining)


Introduction
In January 2020, the World Health Organization (WHO) declared the severe coronavirus disease 2019 (Covid-19) outbreak to be a Public Health Emergency of International Concern 1 . Given the rapid spread of cases across the world, the new coronavirus; Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), has caused several hundred thousand deaths worldwide. Clinical manifestations of Covid-19 such as fever, dry cough, asthenia, headache, myalgia, anosmia, diarrhea and a great recent number of skin manifestations are widely referenced [2][3][4] .
Previous post-mortem lung analysis of SARS-CoV-2 demonstrated in ammation, edema and type II pneumocyte hyperplasia 5 , but many patients had a procoagulant state and/or severe hypoxemia with relatively well-preserved lung mechanics 7 . Based on observed disease patterns, Covid-19 is not just a serious respiratory viral disease, but rather a systemic multi-organ viral invasion with microvascular injury and thrombosis [5][6][7] . However recent data indicates that anticoagulated patients were more likely to require invasive mechanical ventilation with a poor outcome associated 8 . Mechanisms underlying the vascular and tissue impact are not well known yet. Histological data with immunohistochemistry (IHC) and direct immuno uorescent (DIF) techniques in COVID-19 tissues´ patients are still limited and mainly focused on post-mortem analysis. There are barely any studies performed in living tissue of Covid-19 patients.
Since SARS-CoV-2 largely affects cutaneous tissue, skin Covid-19 lesions could provide extensive information. A skin biopsy could be a very accessible procedure to assess the pathophysiologic mechanisms in depth in living tissue of Covid-19 patients. Vast clinical and histological descriptive expressions of skin on Covid-19 patients could provide insightful ndings.
Herein, we aim to examine in depth living tissue of skin lesions related to Covid-19. Immune and in ammatory histologic markers were evaluated in order to help better understand physio-pathological mechanisms underlying Covid-19 infection on living tissue.

Materials And Methods
Five skin lesions from caucasian Covid-19 adult patients were selected for cutaneous tissue histological examination. Skin lesions were manifested during the infection period and they were not associated to any other disease. Skin biopsies were performed under patient consent. Both, punch (case 1, 3 and 5) and

Case Reports
We report ve skin lesions falling under pseudo-chilblain (1) and purpuric (2-5) Covid-19 patterns 4 . Cases 1 to 3 on the ngers, case 4 on the lower limbs and case 5 on the foot of the patients (Description shown on Table 1 and Figure 1).
All patients presented asthenia and gastrointestinal symptoms (abdominal pain, diarrhea, nauseas or vomits) among others ( Table 1). None of them had drug allergies, neither previous history of dermatological disease, acrocyanosis, Raynaud phenomenon, nor perniosis. Distal pulses were preserved. Among relevant data of blood sampling exam, an elevated D-dimer, IL6 and ferritin serum levels, with an increase prothrombin time (PT) were found on cases 1,3 and 4.
Our study identi ed common strong histological results across the ve cases.
Histological common ndings across the cases: Our ndings showed a common pattern on skin lesions as visualized in Table 1. The injury's brunt was mainly focused on vascular tissue. All of them presented a vasculopathic reaction pattern with prominent vascular endothelial and myointimal cell growth ( Interestingly, positive ICH stained for CD31 con rmed a prominent endothelial cell hyperplasia and positive stained SMA provided a moderate myointimal capillary cell hyperplasia (Figure 3. 1a-5a and 1b-5b, respectively). The result was a highly obliterative vascular lumen in all the patients. Additionally, an increased number of replicating vascular cells positive stained for Ki67 and Cyclin D1 antibodies were identi ed (Figure 4. 1a-5a and 1b-5b, respectively). Proliferation was mainly dependent on endothelial cells and myointimal vascular cells.
An extensive signi cant vascular C4d and C3 deposition throughout vascular cell wall was observed (Figure 4. 1c,2c,1d). A moderate granular IgM deposition were manifested. Neither IgG nor IgA deposition was identi ed.
More Covid-19 patients with skin lesions were identi ed and analyzed across this period with same histological results (data nor shown).

Discussion
Our ndings evidence a common cutaneous vasculopathic reaction pattern based on prominent vascular endothelial and myointimal growth across Covid-19 patients. In our study, the lesions observed seem to be associated to an obliterative micro-angiopathy that could be induced by SARS-CoV-2 whose target seems to be the vascular endothelial and myointimal cells with complement activation. To our knowledge, there is no existing previous data that examinates in great detail infected SARS CoV-2 living tissue in order to understand the mechanisms underlying vascular and its surrounding tissue compromise.
It is suspected that Covid-19 patients may be facing a microvascular process with vascular wall cell injury. Coronavirus uses the angiotensin converting enzyme 2 (ACE2) endothelium receptor, as an entry cell point [7][8][9][10] . According to our results, this phenomenon could cause a vasculopathic reaction pattern with a marked regenerative growth of both, vascular endothelial cells and vascular myointimal cells.
Additionally, extensive deposits of the complement complex C4d as well as C3 were manifested and could contribute to this vascular wall alteration.
Our ndings showed that this vascular cell growth and distortion provokes the obliterative capillary lumen observed. It could lead to a secondary tissular non-acute hypoxia with a higher vascular cell growth by a feedback effect. It is highly likely that increased vascular-cell proliferation could be a distinctive characteristic of the early response to hypoxic insult 11 . Vascular cell proliferation is con rmed in our study by the increased numbers of replicating cells positive stained for Ki67 and Cyclin D1 found in both, vascular endothelial cells and myointimal vascular cells and directly involved in cell-cycle progression.
Asthenia is a very common reported Covid-19 symptom. This symptom is also found amongst our patients. This non-acute tissular hypoxia could contribute to explain this symptom.
Ischemic signs on the tissue were not identi ed probably due to a non-acute hypoxia. To the contrary, it was found a regenerative epidermal hyperplasia with tissue structure preservation. Our results agree with previous post-mortem lung studies of SARS-CoV-2, that also demonstrated type II pneumocyte hyperplasia 5 . Therefore, many patients presented a procoagulant state and/or severe hypoxemia with relatively well-preserved organ structure and mechanics. Furthermore, our results indicated that impairment of endothelium crosstalk seems to lead to bloodvascular barrier dysfunction followed by an increased vessel permeability and erythrocyte extravasation. This phenomenon could explain the hemorrhages or ecchymosis-hematomas Covid-19 patients manifested and also seen in other tissues 5 . Previous post-mortem lung studies also suggest that pulmonary edema caused by capillary transudation is a common characteristic of Covid-19 5 .
Microvascular endothelial cell injury could subsequently activate the clotting pathway 7 . Thromboembolic events have been identi ed in patients with Covid-19 6 . Across these patients, anticoagulation treatment is used. However, emergent studies have found a positive correlation between anticoagulation and a greater overall illness severity 8 . According to our results, anticoagulation by itself could not completely reverse vascular lumen obliteration, with consequent hemorrhagic increased risk associated.
Vascular endothelial growth factor (VEGF) is known as the most potent inducing factor to increase angiogenesis and vascular permeability 11 . This, together with activation of the complement, could have an important role in inducing and maintaining Covid-19 vasculopathic reaction pattern. These observations could help nd potential speci c pharmacotherapy for ghting against Covid-19 severe patients.
Our work aims to emphasize, as previously reported 9 , the importance of incorporating living tissue biopsy when examining Covid-19 mechanisms. It could also help to disease diagnose. Since SARS-CoV-2 affects cutaneous tissue too, skin Covid-19 lesions could provide extensive, accessible information and can mirror other organs of the body. Vast clinical and histological descriptive expressions of skin on Covid-19 patients could provide insightful ndings to improve the diagnosis and management of the disease.

Conclusions
Based on our results, Covid-19 could include a microvascular injury consisting on endothelial and myointimal intensive growth with complement activation. This mechanism, together with increased vascular permeability, could contribute to obliterative vascular lumen and hemorrhage in Covid-19. Therefore, activation of the complement and angiogenic pathways could have an important role in inducing and maintaining this vasculopathic reaction pattern. Moreover, anticoagulation treatment might be reviewed. Findings of this study could contribute to a better understanding of physio-pathological mechanisms underlying SARS CoV-2 infection on living tissue and could help further studies nd potential targets for speci c therapeutic interventions in Covid-19 severe patients.

Declarations
Con icts of Interest: The authors have no con icts of interest.     IHC analysis for C31stain (1a-5a) and SMA stain (1b-5b) of live tissue samples from Covid-19 patients.